Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex Published online 3 September 2010 Nucleic Acids Research, 2011, Vol. 39, No. 1 347–358 doi:10.1093/nar/gkq749 Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex 1 2,3,4 1 2,4 Israel S. Ferna ´ ndez , Akio Yamashita , Ernesto Arias-Palomo , Yumi Bamba , 1 5 1 2, Ruben A. Bartolome ´ , M. Angeles Canales , Joaquı´n Teixido ´ , Shigeo Ohno * and 1, Oscar Llorca * Centro de Investigaciones Biolo ´ gicas, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Ramiro 2 3 de Maetzu 9, 28040 Madrid, Spain, Department of Molecular Biology, Department of Microbiology and Molecular Biodefense Research, Yokohama City University School of Medicine, Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan and Dpto. Quı´mica Organica I, Facultad de Ciencias Quımicas, Univ. Complutense, Av. Complutense s/n, 28040 Madrid, Spain Received February 26, 2010; Revised and Accepted August 6, 2010 ABSTRACT SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of SMG-9 is part of a protein kinase complex, SMG1C, IDRs could facilitate the transit of SMG-9 through which consists of the SMG-1 kinase, SMG-8 and several macromolecular complexes. SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and INTRODUCTION targets for degradation mRNAs harboring prema- Eukaryotic gene expression comprises a complex set of ture translation termination codons. Here, we have biochemical reactions starting with the transcription characterized SMG-9, showing that it comprises an of the genetic information and ending in the synthesis of N-terminal 180 residue intrinsically disordered proteins. Between these two events, post-transcriptional region (IDR) followed by a well-folded C-terminal modifications and remodelling are required to assemble domain. Both domains are required for SMG-1 a mature mRNA that can be translated by the ribosome, binding and the integrity of the SMG1C complex, and several surveillance mechanisms ensure the fidelity and accuracy of these processes. Nonsense-mediated whereas the C-terminus is sufficient to interact mRNA decay (NMD) is a post-transcriptional surveil- with SMG-8. In addition, we have found that lance mechanism that, in eukaryotes, recognizes and SMG-9 assembles in vivo into SMG-9:SMG-9 and, degrades mRNAs containing premature translation ter- most likely, SMG-8:SMG-9 complexes that are not mination codons (PTCs) to prevent the accumulation of constituents of SMG1C. SMG-9 self-association is potentially harmful truncated polypeptides encoding for a driven by interactions between the C-terminal truncated protein (1,2). domains and surprisingly, some SMG-9 oligomers The NMD machinery marks a PTC-containing mRNA are completely devoid of SMG-1 and SMG-8. We for degradation through a highly sophisticated sequence propose that SMG-9 has biological functions of protein-protein interactions involving different poly- beyond SMG1C, as part of distinct SMG-9- peptides (2,3). Until recently, seven conserved core containing complexes. Some of these complexes factors for NMD had been identified to be present in most metazoan, SMG-1, Upf1, Upf2, Upf3, SMG-5, may function as intermediates potentially regulating *To whom correspondence should be addressed. Tel: +34 91 837 3112 (extn 4446); Fax: +34 91 536 0432; Email: ollorca@cib.csic.es Correspondence may also be addressed to Shigeo Ohno. Tel: +81 45 787 2596; Fax: +81 45 785 4140; Email: ohnos@med.yokohama-cu.ac.jp Present address: Israel S. Ferna´ ndez, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 348 Nucleic Acids Research, 2011, Vol. 39, No. 1 SMG-6 and SMG-7. Thanks to an intense current complex and probably up-regulates the kinase activity of research effort, a picture of the players and the molecular SMG-1 since SMG-1:SMG-9 complexes show higher mechanisms involved in NMD is starting to emerge (1,2). activity than SMG-1 (12). Both SMG-8 and SMG-9 are Yet, many aspects remain obscure and several models required for mammalian NMD and the stable association have been proposed to explain the molecular mechanisms between these two proteins is an absolute requirement for by which the NMD machinery tags an mRNA for degrad- the inhibition of the NMD response until a genuine PTC is ation (4,5). An interesting debate in current literature recognized (12). upholds a vision of NMD regulated by the outcome of a In order to improve our understanding of the functions competition between stimulating and downregulating and molecular structure of the SMG1C complex, here we signals (3–6). In any case, the topic of what determines have characterized SMG-9. We present evidence showing the fate of a PTC-containing mRNA is still the subject that SMG-9 comprises two distinct domains. The of open research (3,5,7). N-terminal domain is an intrinsically disordered region One of the central players articulating the NMD (IDR) essential for the maintenance of the structural in- response is SMG-1, a large protein of roughly 430 kDa tegrity of the SMG-1:SMG-8:SMG-9 complex. IDRs are a that belongs to the phosphatidylinositol 3-kinase-related hot topic of research, as they appear to provide the protein kinase (PIKK) family of serine-threonine kinases essential specificity and malleability for the large macro- (8,9). SMG-1 is a component of mRNA surveillance molecular machines requiring multiple and variable complexes and the phosphorylation of Upf1 by SMG-1 protein–protein interactions, as those functioning in all is the single essential event in metazoans to trigger all stages of eukaryotic gene expression (19,20). Unexpec- the latter processes leading to the degradation of an tedly, several experiments in vivo reveal that, besides is mRNA (10). A complex known as SURF participation as a component of the SMG1C complex, (SMG-1:Upf1:eRF1:eRF3) and containing SMG-1, Upf1 SMG-9 can assembly as homodimers and, most likely, and the eukaryotic release factors eRF1 and eRF3 is SMG-8:SMG-9 heterodimers that could represent inter- assembled on a termination codon together with the mediates regulating the assembly of the SMG1C complex. ribosome (11–13). The ribosome:SURF complex can interact with a downstream exon–junction-complex MATERIALS AND METHODS (EJC), a protein complex deposited 20–24 nt upstream the exon–exon junction, through the Upf2 and Upf3 Prediction of ordered and disordered regions proteins, activating the kinase activity of SMG-1 on We analysed the predicted ordered and disordered regions Upf1. Phospho-Upf1 is thought to recruit the in the sequences of SMG-1, SMG-8 and SMG-9 using one mRNA-decapping as well as the RNA-degrading machin- of the most accepted predictors of naturally disordered ery that eventually degrades the mRNA containing the regions, PONDR (http://www.pondr.com) (21). The PTC (2,3). default predictor VL-XT was used. SMG-1 has been shown to play other roles besides controlling NMD. Depletion of SMG-1 in human cells 12–180 Cloning, expression and purification of NT-SMG-9 influences the response to DNA damage (8,14) and regu- lates the association of telomeric repeat-containing RNA NT-SMG-9 cDNA was subcloned between the EcoRI and at telomeres (15). SMG-1 is required for adequate regula- NcoI sites on modified N-terminal HisTag pRAT4, pRHO tion of p53 phosphorylation upon genotoxic and oxidative and pGEX-6P-2 plasmids (GE Healthcare Bio-Sciences, stress and controls cell proliferation and apoptosis Buckinghamshire, UK). The initial GST fusion construct (14,16–18). Although the molecular bases of all these was made comprising amino acids 1–180. After the obser- vation of spontaneous self-cleavage in the initial processes are unclear, many of these functional features GST-fusion construct, the site of cleavage was identified parallel those of other PIKKs, suggesting some cooper- by mass spectroscopy and the construct re-cloned ation among the members of this family of kinases (8,13). comprising amino acids 12–180. All proteins were ex- Very recently, two novel components of a SMG-1 pressed in Escherichia coli strain BL21(DE3) and the ex- complex have been discovered and named SMG-8 and SMG-9 (12). These proteins were isolated due to their pression detected in a soluble fraction after cell lysis by co-purification with SMG-1, with which they form a sonication. GST-fusion proteins were puriEed with stable complex (SMG-1:SMG-8:SMG-9), named GST-Trap columns (20 ml, GE Healthcare Bio-Sciences) SMG1C. SMG-8 and SMG-9 are tightly associated with and NT-SMG-9 was isolated from GST after digestion SMG-1 and they seem to regulate its kinase activity and with the 3C protease. A second GST-trap column the remodelling of the mRNA surveillance complex. followed by a cation exchange chromatography in Interestingly, additional NMD factors, Ruvbl1, Ruvbl2, SP-sepharose HiTrap colum (5 ml, GE Healthcare RPB5 and SMG-10, have just been described, highlighting Bio-Sciences) followed by a final step of gel-Eltration the complexity of the NMD machinery (13). SMG-8 is a chromatography (Sephacryl S-100, GE Healthcare 991 amino acid protein which has been proposed to Bio-Sciences) yielded a highly pure preparation as regulate the correct localization of SMG-1 at the judged by SDS–PAGE after Coomassie brilliant blue PTC-stalled ribosome to form the SURF complex (12). staining. Protein concentration was determined by UV SMG-9 is a 520 amino acid protein comprising a central absorption at 280 nm. The protein solutions were putative nucleotide-triphosphatase domain. SMG-9 seems concentrated with an Amicon Ultra device (Millipore, to regulate the formation of the SMG-1:SMG-8:SMG-9 Bedford, MA, USA). Mass spectroscopy was used to Nucleic Acids Research, 2011, Vol. 39, No. 1 349 assess the identity as well as the purity of final The soluble fractions were pre-cleared with sepharose 4B preparations. (Sigma) and then incubated with streptavidin–sepharose (GE Biotech) for 2 h at 4 C with gentle rotation. Spectroscopic techniques Pre-cleared lysates were incubated with streptavidin– sepharose or anti-V5 antibodies for 2 h or 1 h at 4 C Circular dichroism (CD) spectra were recorded on a with gentle rotation. For antibodies, subsequently, the JASCO J-805 spectropolarimeter. An optical cuvette soluble fractions were incubated with 30 ml of protein G with a 1-mm path length was used. The temperature of sepharose (GE Biotech) for an additional 1 h at 4 C with the measuring cell was maintained at 25 C. Spectra were gentle rotation. After washing with RNase() T-lysis collected in a spectral range of 200–250 nm with a buffer, the affinity-purified protein complexes were path-length of 1 nm. The NT-SMG-9 preparation was eluted by incubation at 4 C for 30 min with RNase() dissolved in 20 mM sodium phosphate (pH 7.2) and lysis buffer containing 2 mM biotin (Sigma) or SDS 50 mM NaCl at a concentration of 15 mM. Data was sample buffer, respectively. All proteins in western blot analysed using the software KD2. experiments were detected with an ECL western blot Fluorescence spectra were acquired on an F-4500 detection kit (GE Biotech) or Lumi-Light (Roche). All Fuorescence spectrophotometer (Hitachi, Tokyo, Japan) experiments were performed two to three times, and at 25 C. The concentration of NT-SMG-9 was 15 mM. typical results are shown. Buffer solution contained 20 mM sodium phosphate (pH 7.2) and 50 mM NaCl. The excitation wavelength Size exclusion chromatography of SMG-9 complexes was 295 nm, and the emission spectra were recorded between 285 and 500 nm. Denaturing conditions For the preparation of HeLa cell extracts for gel column involved the measurement with the same conditions fractionation, 3  10 HeLa cells were re-suspended in an buffer and protein concentration but in the presence of equal volume of lysis buffer containing 10 mM Tris–HCl 6 M guanidinium hydrochloride (Pierce). at pH 8.0, 150 mM NaCl, 0.4% NP40, 2 mM MgCl , In the NMR spectroscopy experiments, samples for H 0.2 mM DTT, 0.1 mM PMSF, 100 nM Okadaic acid and monodimensional spectra were prepared in 20 mM sodium 200 mg/ml RNaseA. After incubation on ice for 10 min, phosphate (pH 7.2) and 50 mM NaCl at a concentration cells were lysed by 15 hand-strokes of a loose-fit of 100 mM. N-labelled samples were prepared in M9 Potter-Elvehjem homogenizer. The cell lysate was medium at a concentration of 200 mM. The NMR centrifuged at 15 000g for 30 min, and the supernatant samples contained 10% D O.The monodimensional as was loaded onto a 24 ml Superose 6 FPLC column (GE 1 15 well as H– N heteronuclear single-quantum correlation Biotech) equilibrated with lysis buffer. Fractions (400 ml) (HSQC) spectra were acquired at 25 C on a Bruker were collected from 5 to 24 ml elution volume. Fractions DMX-600 spectrometer equipped with a cryoprobe. were pooled and concentrated. Proteins were detected by western blotting. A control molecular marker was For reagents (antibodies) obtained by running the proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), Anti-SMG-8 and -SMG-1 have been described earlier aldolase (158 kDa), and RNaseA (15 kDa) (GE Biotech) [Yamashita et al. (12)]. Anti-HA (clone 3F10) (Roche), on the same column under the same conditions. anti-SBP (SantaCruz), anti-mTOR (Cell Signaling Technology), anti-aPKC (C-20) (SantaCruz) were obtained commercially. RESULTS Affinity purification, immunoprecipitation and SMG-9 comprises two distinct structural domains western blot analysis The first description of SMG-9 reported the presence of a pEF_Flag-HA-SBP-SMG-9 (2–520) (for Figure 5B), putative nucleotide-triphosphatase domain comprising pEF_Flag-HA-SBP-NT-SMG-9 (2–181), pEF_Flag- residues 181–520 (12). We now performed a more HA-SBP-CT1-SMG-9 (185–520), pEF_Flag-HA-SBP- thorough analysis of the SMG-9 sequence using several CT2-SMG-9 (175–520), pcDNA5/NTAP(CBP-SBP)- bioinformatic methods, which revealed the presence of SMG-9 (2–520) (for Figure 5C), pSR_Strep-HA-SMG-9 an unusual N-terminal region. The N-terminal region of full (2–520), pSR-V5-SMG-9 full (2–520), pSR_V5- SMG-9 was enriched in prolines, polar and charged NT-SMG-9 (2–181) and pSR-V5-CT-SMG-9 (182–520) residues while showing a low content in the hydrophobic were constructed by cloning each cDNA fragment by residues that most frequently form the hydrophobic core standard methods. siLentGene-puro-siSMG-9UTR of conventional protein domains (Supplementary Figure (siRNA sequence targeted to the 3 -UTR of the SMG-9 S1). These features are the signature of intrinsically dis- mRNA: GGAGAGGAATGTCATGCAC) was con- ordered regions (IDRs), segments of proteins that under structed by method described in manual. native conditions do not fall into a conventional fold and A 293T cells were transfected using HEKfectin which participate in specific binding to targets in highly (Biorad), and lysed with a loose-fit Potter–Elvehjem hom- complex multi-component macromolecular machines (19). ogenizer in T-buffer [20 mM HEPES–NaOH at pH 7.5, We searched for the presence of IDRs in SMG-9 based on 50 mM NaCl, 0.05% Tween-20, 2.5 mM MgCl , 0.5 mM the distinctive signature of their sequence composition and DTT, protease inhibitor cocktail (Roche), phosphatase in- conservation of amino acids. Several in silico disordered hibitor cocktail (Roche) and 100 mg/ml RNaseA (Qiagen)]. predictors have been developed, all of them showing 350 Nucleic Acids Research, 2011, Vol. 39, No. 1 1 180 181 520 higher proportion of hydrophobic residues (Figure 1C). Also, a comparison of the ratio between hydrophobicity versus net charge in several proteins placed the NT-SMG- NT-SMG-9 CT-SMG-9 (NTPs-like) 9 domain well within the group of other known intrinsic- 1.0 ally unstructured proteins whereas the C-terminal domain showed the characteristic pattern of folded domains 0.8 (Supplementary Figure S2). 0.6 0.5 0.4 The recombinant N-terminus of SMG-9 is a 20 kDa soluble monomeric domain 0.2 To further analyse the biophysical and structural 0.0 properties of the NT-SMG-9 domain, several milligrams 0 100 200 300 400 500 of highly pure protein were required, only achievable by Residue Number recombinant production. Non-specific degradation of par- tially folded heterologous proteins expressed in BC NT-SMG-9 CT-SMG-9 Escherichia coli is a common problem, which could be potentially enhanced in the case of an IDR, due to its intrinsic disordered structure. To increase the chances of success in the production of NT-SMG-9 in E. coli, we set up three parallel strategies taking advantage of this well-established prokaryotic system. We cloned the cDNA corresponding to the first 180 residues of SMG-9 (NT-SMG-9) into three different expression plasmids con- Polar Charged Hydroph. Polar Charged Hydroph. taining either (i) a N-terminal hexahistidine tag (HisTag), Figure 1. Analysis of SMG-9 primary structure. (A) PONDR analysis (ii) a N-terminal OmpA peptide inducing the secretion of of the SMG-9 amino acid sequence. A PONDR score >0.5 predicts the expressed protein to the periplasmic space and a those amino acids belonging to a disordered region. A stretch of dis- ordered amino acids of more than 50 residues are usually considered a C-terminal HisTag and (iii) a fusion protein with a gluta- disordered domain. This analysis revealed that the sequence of SMG-9 thione synthetase transferase (GST) and a site for the 3C could be divided in two regions: an N-terminal 180 residue intrinsically protease between the GST and the target construct to disordered region and a C-terminal folded domain, encoding a putative remove the tag. We found no expression when using the nucleotide-triphosphatase (NTPase)-like domain (12). Distribution of N-terminal HisTag, whereas a small secretion of the ex- types of amino acid in NT-SMG-9 (B) and the C-terminal region of SMG-9 (C). NT-SMG-9 revealed a propensity towards polar and pressed protein was observed with the periplasmic charged amino acids while the C-terminal domain was enriched in secretion-inducing vector, albeit with a high degree of hydrophobic residues. Unstructured domains frequently exhibit a low non-specific degradation (not shown, see below and content in hydrophobic amino acids and a bias towards charged and 12–180 Figure 2A for the purification of NT-SMG-9 ). The polar residues. best results were obtained with the fusion construct con- taining a GST tag. In standard conditions, GST-NT- SMG-9 was expressed as a soluble protein, but 20–30% excellent predictive value (19,20). We used PONDR of the recovered protein from the GST column was (‘Predictor OfNatural Disordered Regions’) non-specifically proteolysed. We identified the non-specific (http://www.pondr.com/) (21) to look for potential dis- cleavage site by sequencing of the N-terminus and by ordered domains in SMG-9. SMG-9 exhibited two mass spectrometry of the spontaneous truncated clearly distinct regions in its primary structure product. We found that the first 11 amino acids of (Figure 1A). A C-terminal region comprising residues NT-SMG-9 were removed non-specifically during purifi- 181–520 was predicted to conform to a conventional cation and we therefore recloned the fragment with- well-folded domain in agreement with its description as out these residues in the GST vector to produce a a putative NTPase domain based on sequence homology more stable product. This construct, GST-NT-SMG- 12–180 (CT-SMG-9 from now on). In contrast, PONDR pre- 9 , missing the first 11 amino acids, expressed as a dicted a large highly disordered region encompassing the soluble protein and was entirely stable after perform- first 180 residues of SMG-9 (NT-SMG-9 from now on) ing the purification steps described earlier (Figure 2A). (Figure 1A). The final steps during the purification protocol Disordered regions typically exhibit a higher ratio involved a second GST affinity column to remove the between the sum of polar plus charged amino acids and GST-tag, an intermediate step of cationic exchange the hydrophobic residues than structured folded domains, and a final gel filtration chromatography (Figure 2B). since they do not form a conventional hydrophobic core. This protocol allowed the efficient production of soluble 12–180 An analysis of the distribution of different types of amino NT-SMG-9 with a yield of 3–4 mg of protein per acids in SMG-9 revealed a propensity of NT-SMG-9 liter of medium (Figure 2C). towards polar and charged amino acids (Figure 1B), We characterized the hydrodynamic behaviour of 12–180 whereas the C-terminal domain of SMG-9 showed a E. coli expressed NT-SMG-9 protein in solution to Percentage PONDR Score Order Disorder Nucleic Acids Research, 2011, Vol. 39, No. 1 351 BC 12–180 Figure 2. Expression and purification of NT-SMG-9 in E. coli.(A) SDS–PAGE of three different constructs assayed to produce a soluble fragment of NT-SMG-9. Left, N-terminal hexahistidine tag (pRHT vector); middle, periplasmic secretion vector (pRHO vector); and right, GST fusion protein (pGEX-6p-2 vector). The total cell extract right before induction (column T0), 5 h after induction with 1 mM IPTG (column T5) and the soluble fraction after sonication of the T5 sample (column S), are shown. For the periplasmic construct the supernatant (column SN) of the culture of T5 is shown, since the periplasmic space of E .coli is very leaky, over-expressed proteins can be easily localized in the supernatant of the centrifuged culture. Lastly, in those two constructs where some expression was detected, the elution from an in-batch incubation of the supernatant (periplasmic secretion construction) or the soluble fraction of T5 (GST-fusion construction) with HisTrap resin or GST–sepharose resin (column O), 12–180 are shown. The pull-down protein in the GST-NT-SMG-9 construct is labelled. (B) SDS–PAGE of a purified NT-SMG-9 after the first GST-trap column before (column NC) and after a 4 h digestion (column C) with 3C protease. (C) Final preparation after applying the purification protocol described in the text. define its state of aggregation using analytical gel filtration performed through sedimentation velocity experiments and analytical ultracentrifugation (Supplementary Figure (Supplementary Figure S3B) unambiguously showed that 12–180 12–180 S3). NT-SMG-9 eluted as a single sharp peak in size NT-SMG-9 behaved as a single species in solution, exclusion chromatography, with a retention volume of with a Svedberg coefficient of 1.1 S corresponding to a 1.72 ml, compatible with a molecular mass of 20 kDa molecular weight of 19.5 ± 0.4 kDa (Supplementary after calibration of the column (Supplementary Figure Figure S3C). These results were further confirmed by sedi- S3A). Less than 5% of the protein eluted as a large aggre- mentation equilibrium analysis performed at two different 12–180 gate in the void volume, indicating that NT-SMG-9 velocities, which notably agreed well with the previous behaved as expected for a single monomeric species in data (Supplementary Figure S3D). solution. This experiment was performed in medium-high Taking all the hydrodynamic data into account, we 12–180 ionic strength conditions (300 mM NaCl) that were conclude that the NT-SMG-9 domain did not form required to avoid interaction of the protein with the any major aggregate, the predominant species in solution column matrix. In addition, ultracentrifugation ana- being a monomer, at medium-high as well as low ionic lysis at lower ionic strength conditions (50 mM NaCl) strength conditions. 352 Nucleic Acids Research, 2011, Vol. 39, No. 1 to zero in the vicinity of 222 nm, the CD-spectra of 12–180 NT-SMG-9 showed a minimum at 205 nm and negative values of ellipticity from 235 to 200 nm (Figure 3A). This suggested the presence of certain content in secondary structure. In addition, fluorescence spectra with an excitation wavelength of 295 nm revealed a significant increase in the fluorescence emission upon de- 12–180 naturation of NT-SMG-9 using 6 M guanidinium hydrochloride compared to native conditions (Figure 3B), strongly suggesting the presence of a certain degree of secondary structure quenching the fluorescence 12–180 of the two tryptophans of NT-SMG-9 in the native protein. NMR spectroscopy was performed to definitively deter- mine the presence of an IDR at the N-terminus of SMG-9. 1 12–180 A H 1D spectrum of NT-SMG-9 showed two groups of signals, a first group of thin, well-resolved peaks (6.5–8 ppm) and a group of superposed signals accumulating between 8 and 8.5 ppm (Figure 4A). This spectrum would be compatible with an unfolded polypep- tide, being the first group of sharp peaks those signals corresponding to the flexible lateral side chains and the aromatic protons whereas the N–H backbone would appear as those signals between 8 and 8.5 ppm. We also 1 15 performed H- N-HSQC 2D experiments after labelling 12–180 15 NT-SMG-9 with N (Figure 4B), and we found that the majority of the signals attributable to the N-H backbone overlapped within a very narrow H chemical shift, ranging from 7.75 to 8.5 ppm. This is a typical spectrum for intrinsically disordered regions, where defined signals (in contrast to aggregated protein) are concentrated in a narrow range (in contrast to folded proteins). Signals of the two tryptophans present in 12–180 NT-SMG-9 were detected at 10.25, 128.89 ppm and 12–180 Figure 3. Spectroscopic analyses of NT-SMG-9 .(A) Far 1 15 12–180 10.1, 127.48 ppm, H, N chemical shifts, respectively. UV-circular dichroism spectrum of NT-SMG-9 .(B) Fluorescence 12–180 spectra of NT-SMG-9 in 50 mM phosphate buffer and 50 mM These chemical shifts are characteristic of solvent NaCl (black circles) and in the same buffer but in the presence of exposed tryptophan residues as the amino acids of a dis- 6 M Guanidinium hydrochloride (white circles). ordered protein. In addition, the HSQC spectrum showed several well-dispersed peaks typical of a folded structure, suggesting the presence of some residual structure as pre- viously suggested by CD and fluorescence spectroscopy The N-terminal domain of SMG-9 is an intrinsically data. disordered region Recent studies suggest that IDRs do not show uniform The N- and C-terminal domains of SMG-9 are required structural properties, but their structure ranges from a to maintain the integrity of the SMG1C complex fully unstructured protein (‘random coils’) to partially structured regions (‘pre-molten globule’) and more We examined the relevance of the two domains of SMG-9 ‘folded’ proteins containing some elements of secondary to maintain the integrity of the SMG-1:SMG-8:SMG-9 structure (‘molten globule’) (19,20). Proteins in the first complex in cells. For this purpose, we expressed group show no secondary structure at all as well as hydro- full-length SMG-9 and fragments comprising the 2–181 dynamic dimensions like those of coiled-coils. In contrast, N-terminal (NT-SMG-9 ) and C-terminal (CT-SMG- 185–520 pre-molten globules present a core of secondary structure, 9 ) domains as SBP (Streptavidin Binding Peptide) although less dense than that found in structured proteins. tagged fusion proteins in 293T cells. To avoid the inter- To gain insight into the structural properties of NT-SMG- ference of endogenous SMG-9, this protein was down 12–180 9 we investigated its secondary structure content regulated using RNA interference targeted to using two spectroscopic techniques, ultraviolet-circular di- 3 -untranslated region of SMG-9. The expressed proteins chroism (UV–CD) and fluorescence spectroscopy were bound to Streptavidin-beads in presence of RNaseA, (Figure 3). Whereas completely unfolded polypeptides to remove interactions mediated by RNA, eluted and the are characterized by a well-defined CD spectrum with a presence of SMG-1 and SMG-8 in the pull-downed minimum in the vicinity of 200 nm and an ellipticity close material tested by western blotting (Figure 5B). Whereas Nucleic Acids Research, 2011, Vol. 39, No. 1 353 10 86420 H ppm 10.00 9.50 9.00 8.50 8.00 7.50 7.00 H ppm 12–180 1 12–180 Figure 4. NMR spectroscopy analysis of NT-SMG-9 .(A) H monodimensional spectrum of NT-SMG-9 showing the overlapping of 1 15 15 12–180 signals in a narrow chemical shift in a range centered at 8.25 ppm. (B) H- N HSQC spectrum of N-labelled NT-SMG-9 unambiguously identified this domain as inherently unstructured due to the absence of well dispersed cross peaks. The presence of well-resolved peaks and the absence of dispersion discarded any non-specific aggregation. each product was adequately expressed, only full-length of SMG-8 whereas contributions of the N- and C-terminal SMG-9 co-purified with SMG-1 and SMG-8 in presence domains of SMG-9 are required to bind SMG-1. of RNaseA. These experiments indicated that both the N- The experiment described earlier was performed after and C-terminal domains of SMG-9 are necessary for the simultaneous co-transfection of full-length HA-tagged integrity of the SMG1C complex. To map the requirement SMG-9 and the SBP-tagged constructs, and surprisingly of SMG-9 for these interactions more precisely, in a the pull-downs revealed that the full-length and two separate set of experiments, we simultaneously tested C-terminal constructs of SMG-9 tested were interacting with full-length HA-SMG-9. Unexpectedly, CT-SMG- two C-terminal constructs comprising residues 185–520 185–520 175–520 185–520 (CT-SMG-9 ) and 175–520 (CT-SMG-9 ) 9 was forming a tight complex with full-length tagged with SBP. SBP-pull-downs confirmed that SMG-9, devoid of SMG-1 and SMG-8, in striking 185–520 CT-SMG-9 was not capable of recognizing contrast with the behaviour of full length SMG-9 that 175–520 SMG-1 or SMG-8. Interestingly, CT-SMG-9 , reproducibly pulls downs a significant amount of where a small N-terminal segment flanking the SMG-1 and SMG-8 (Figure 5C). In addition, full length C-terminal domain was incorporated, was sufficient to SBP-SMG-9 was found to interact with full length recognize SMG-8 at a similar level than full-length HA-SMG-9, indicating that the association between SMG-9 (Figure 5C), whereas the recognition of SMG-1 SMG-9 molecules takes place also in the context of the was heavily impaired. These results strongly suggested full protein (Figure 5C). Hence, the existence of putative that CT-SMG-9 is directly responsible for the recognition SMG-9 oligomers was further investigated. N ppm 354 Nucleic Acids Research, 2011, Vol. 39, No. 1 A B SBP-SMG-9 1 180181 520 SMG-9 IDR NTPs-like 2 520 SBP (SMG-9) 185 520 175 520 SMG-1 2 520 SMG-8 2 520 SMG-1 182 520 SMG-8 SBP-SMG-9 V5-SMG-9 CD SBP (SMG-9) V5 (SMG-9) SMG-1 SMG-8 HA (SMG-9) 185-520 SBP (SMG-9 ) SMG-1 185-520 SBP (SMG-9 ) SMG-8 HA (SMG-9) Figure 5. Effect of NT-SMG-9 and CT-SMG-9 truncation in SMG1C assembly. (A) Schematic structures of SMG-9 construct. (B) 293T cells were transfected with the SMG-9 plasmids shown above together with plasmid expressing the siRNA targeted to 3 -UTR of SMG-9. (C) 293T cells were transfected with the SBP-tagged-SMG-9 plasmids shown above together with the HA-tagged-SMG-9 plasmid. The cells were lysed and pull downed with the streptavidn sepharose in presence of RNaseA. Pull downed products or cell lysates (input) were then probed with the antibodies shown on 185–520 the right. (D) 293T cells were transfected with the V5-tagged-SMG-9 plasmids shown above together with the SBP-tagged-CT-SMG-9 plasmid. The cells were lysed and immunoprecipitated with anti-V5 antibodies in presence of RNaseA. Immunoprecipitated products or cell lysates (input) were then probed with the antibodies shown on the right. ‘Vector’ indicates an empty vector. SMG-9 assembles into stable oligomers with SMG-9 strongly contributes to its self-association (Figure 5D). (homo-oligomers) and SMG-8 (hetero-oligomers) that are However, we failed to detect binding between NT-SMG- not part of SMG1C 2–181 185–520 9 and CT-SMG-9 (Figure 5D) and between 2–181 182–520 2–181 V5-tagged NT-SMG-9 , or CT-SMG-9 or full V5-tagged NT-SMG-9 and SBP-tagged NT-SMG- 2–181 length SMG-9 were co-expressed with SBP-tagged 9 (data not shown). These results correlate with the 185–520 12–180 CT-SMG-9 in 293T cells (Figure 5D). Pull downs finding that recombinant NT-SMG-9 behaved as a by V5 antibody revealed a significant interaction between monomer (Supplementary Figure S3). 182–520 V5-tagged CT-SMG-9 and SBP-tagged CT-SMG- The above experiments implied that SMG-9 could 185–520 9 , indicating that the C-terminal region of SMG-9 assemble other complexes besides SMG1C and we Input SBP-pull down (RNase+) V5- HA- SBP-tagged vector 2-520 175-520 185-520 Input SBP-pull down (RNase+) Input IP:V5 antibody (RNase+) vector vector 2-181 2-520 182-520 2-181 185-520 2-520 Nucleic Acids Research, 2011, Vol. 39, No. 1 355 sought a further confirmation by partially resolving the interaction of SMG-9 with SMG-1. On the other SMG-9 complexes by size exclusion chromatography hand, SMG-9 was found to interact with SMG-8 mostly (Figure 6). HeLa cell extracts were fractionated by gel fil- through its C-terminal domain. We have purified a protein comprising the N-terminal tration and the fractions analysed by denaturing electro- domain and several biophysical approaches (gel filtration phoresis and western blotting. As controls, we used chromatography, analytical ultracentrifugation, CD, and markers of molecular weight (Figure 6A, top line), UV-spectroscopy) have confirmed unambiguously that mTOR, a PIKK member that migrates as a monomer this region behaves as a compact 20 kDa domain with (290 kDa) and as a 0.7–0.8 MDa multi-protein complex the paradigmatic characteristics of unstructured domains (22), and aPKC (78 kDa). In addition to a well-resolved as well as a limited presence of secondary structure. peak corresponding to SMG1C and comprising SMG-1, Whereas misfolded proteins usually aggregate due to the SMG-8 and SMG-9, SMG-9 was detected in two add- exposure of the hydrophobic residues that form the core itional peaks. One, composed only of SMG-9 (monomer, of folded domains, an intrinsically disordered protein is 60 kDa) clearly migrated as a homo-oligomer rather soluble even in the presence of low or no secondary struc- than a monomer, and the apparent molecular weight ture due to the unusual composition of their sequences, correlated with the dimeric species previously detected enriched in polar and charged residues (19,20). The results by pull-down assays. In addition, a second peak contain- obtained by NMR spectroscopy represent the formal ing SMG-9 migrated as a larger complex, which exactly proof that the N-terminus of SMG-9 is an IDR. The ‘sig- co-migrated with SMG-8 as 400 kDa complexes, a nature’ of the mono and bi-dimensional spectra of strong indication of an SMG-8:SMG-9 complex. These NT-SMG-9 is that typical of this group of proteins with results suggested that SMG-9 has biological functions defined signals concentrated in a narrow range (19,20). beyond SMG1C, also maybe regulating SMG1C Furthermore, the combination of NMR data and the spec- assembly by means of several SMG-9-containing troscopy studies suggests that the conformation of the sub-complexes. NT-SMG-9 domain most likely fits into the category of Accordingly, we found that interfering with SMG-9 by intrinsically unstructured proteins termed ‘pre-molten expressing NT-SMG-9 and CT-SMG-9 truncated globules’, where a limited degree of secondary structure products, affected the normal response of cells to could be localized. genotoxic stress and increased susceptibility to apoptosis There is a growing interest in the functional roles of (Supplementary Figure S4 and Supplementary Data), in intrinsically disordered regions and intrinsically dis- agreement with the role described for these complexes in ordered proteins, since these seem to play important genome stability and apoptosis (3,13). HEK-293 cells roles in cellular functions such as transcription regulation, were transfected with vectors coding for full length genome surveillance, chromatin remodeling or mRNA SMG-9, NT-SMG-9 or CT-SMG-9 fragments (see processing (19,20). Algorithms designed to detect these Supplementary Data for details) and tested for suscepti- domains in the primary structure of proteins suggests bility to cisplatin, a DNA alkylating agent known to that the number and functional relevance of IDRs cause cell apoptosis. Cis-platin treatment led to a increase with the complexity of the organism. It has dose-dependent increase in cell apoptosis that was of been estimated that 25% of the total number of proteins higher extent in cells over-expressing the N-terminal or in complex eukaryotic genomes may be totally disordered C-terminal domains of SMG-9 than in cells expressing and 50% could contain at least one disordered region. the full length protein or than in mock cells. This effect IDRs seem to be adequately suited for protein–protein was associated with reduction in pro-caspase-3 levels and interactions in large macromolecular machines involving with increased processing of PARP-1. very specific but transient interactions. These domains seem to provide high specificity sustained in a large area of contact but moderate affinities facilitating interchange DISCUSSION of partners. A recent description of the interaction The activities of the SMG1C complex, containing SMG-1, between Upf1 and the C-terminal domain of Upf2 SMG-8 and SMG-9 are essential for NMD in mammals revealed that this domain of Upf2 is intrinsically dis- (12). SMG-8 and SMG-9 regulate the kinase activity of ordered and the elements of secondary structure are only SMG-1, and SMG-8 is also required to recruit SMG-1 to co-folded upon recognition of Upf1 (23). Given the com- the mRNA surveillance complex. It has been proposed plexity of NMD and more generally of the mRNA pro- that the SMG1C complex could control NMD by inhibit- cessing machinery, intrinsically disordered regions, as the ing SMG-1-mediated Upf1 phosphorylation until a one presently described for SMG-9, could be present in PTC-containing mRNA is properly recognized (12). other components of mRNA processing pathways. Here, we show that SMG-9 comprises an N-terminal We have found that SMG-9 assembles into several domain with the characteristic features of the so-called complexes apart from SMG1C, SMG-9:SMG-9 intrinsically disordered regions (IDRs) and a well-folded complexes, and most likely also SMG-8:SMG-9 C-terminal domain (12). We demonstrate that both the complexes. The finding that SMG-9 dimers could be N-terminal IDR and the C-terminal domain of SMG-9 isolated by gel filtration (Figure 6) and that a partially 185–520 are required for the integrity of the SMG1C complex. truncated dimer (CT-SMG-9 :SMG-9) is completely Both domains are implicated in SMG-1 binding, since free of SMG-1 and SMG-8 (Figure 5C) suggests that removal of either domain disrupts, totally or partially, SMG-9 dimers are not a component of the SMG1C 356 Nucleic Acids Research, 2011, Vol. 39, No. 1 molecular weight (kDa): 669 440 232 158 15 0.5 frac.# 120 40 60 molecular weight (kDa) 669 440 232 158 SMG-1 SMG-8 SMG-9 SMG-1:SMG-8:SMG-9 SMG-8:SMG-9 SMG-9 complex complex dimer mTOR aPKCλ frac.# 25 30 35 40 45 Figure 6. Several SMG-9-containing complexes can be isolated. (A and B) Size exclusion chromatography of SMG-1, SMG-8 and SMG-9 containing complexes. Fractions were run in SDS gels and the presence of either protein tested by western blot using anti-bodies specific to each component. As molecular weight markers, commercial markers (location of their elution peaks indicated in top line), mTOR (290 kDa and 0.7 MDa) and aPKC (78 kDa) were used. complex. This finding is in striking contrast to the could function as intermediaries mediating the assembly of well-characterized behaviour of SMG-9, which pull SMG1C. One possibility could be that the self-association downs SMG-1 and SMG-8 indicating that SMG-9 is a between SMG-9 molecules through the C-terminal component of SMG1C (12). On the other hand, the de- domains, could repress the interaction with SMG-8 and/ tection of SMG-8:SMG-9 complexes indicate that an as- or SMG-1, as proposed for other signalling molecules sociation between these two proteins may also regulate the (Figure 7). For instance, ATM, a member of the PIKK interaction with SMG-1 and the assembly of SMG1C. family of kinases, is activated by a transition from an in- We propose that several SMG-9 containing complexes hibited dimer under normal conditions converting into an that do not contain SMG-1 could potentially have bio- active monomer after DNA damage, a transition requiring logical functions apart from SMG1C. Thus, SMG-1 has phosphorylation of Ser1981 (24). A possible regulatory been shown to participate in the cellular stress response event driving the transitions between the homo-oligomeric (14,16–18), and we find that the expression of truncated and SMG1C-assembled states of SMG-9 could be phos- versions of SMG-9 increased the susceptibility to apoptosis phorylation. Several conserved residues of SMG-9 located (Supplementary Figure S4). In addition, we speculate that within the N-terminal disordered and the C-terminal SMG-9:SMG-9 and, probably, SMG-8:SMG-9 complexes domains are specifically phosphorylated by SMG-1 (12) absorbance 280nM Nucleic Acids Research, 2011, Vol. 39, No. 1 357 Figure 7. Model for the assembly of SMG-9-containing complexes. (Akio Yamashita and Shigeo Ohno unpublished data). Autonomous Region of Madrid (CAM S-BIO-0214-2006 Under certain circumstances, SMG-9 would be activated, to O.L.); Human Frontiers Science Program (RGP39/ ´ ´ interacting with SMG-8 by means of its C-terminal 2008 to O.L.); ‘Consejerıa de Educaciondela domain. This SMG-8:SMG-9 complex could be the Comunidad de Madrid y Fondo Social Europeo’ (to building block to assemble SMG1C. At this stage, we E.A.P.); Japan Society for the Promotion of Science (to cannot completely rule out that SMG-9 could also be a A.Y. and S.O.); Japan Science and Technology dimer within SMG1C, although current data favours a Corporation (to A.Y. and S.O.); Ministry of Education, model with an equimolar SMG-1:SMG-8:SMG-9 Culture, Sports, Science and Technology of Japan (to complex (12). An assembly pathway of SMG1C regulated S.O.); Yokohama Foundation for Advancement of at the level of SMG-9 and SMG-8 would allow the tuning Medical Science (to A.Y.). Funding for open access of the biological functions of SMG1C with the rest of the charge: Spanish Ministry of Science and Innovation. NMD machinery to either restrain or promote the activa- Conflict of interest statement. None declared. tion of SMG-1. The N-terminal disordered domain of SMG-9 participates in the recognition of SMG-1, and it would be the characteristic malleability of IDRs an REFERENCES adequate structural property to allow the transit of SMG-9 through these distinct macromolecular complexes. 1. Conti,E. and Izaurralde,E. (2005) Nonsense-mediated mRNA SMG-9 could therefore appear as an important controller decay: molecular insights and mechanistic variations across species. Curr. Opin. Cell Biol., 17, 316–325. of SMG-1 by regulating the formation of SMG1C and 2. Isken,O. and Maquat,L.E. (2008) The multiple lives of NMD consequently the activation of its kinase activity towards factors: balancing roles in gene and genome regulation. Upf1(12). Nat. Rev. Genet, 9, 699–712. 3. Nicholson,P., Yepiskoposyan,H., Metze,S., Zamudio Orozco,R., Kleinschmidt,N. and Muhlemann,O. (2009) Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions SUPPLEMENTARY DATA beyond quality control and the double-life of NMD factors. Supplementary Data are available at NAR Online. Cell Mol Life Sci., 67, 677–700. 4. Brogna,S. and Wen,J. (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. Nat. Struct. Mol. Biol., 16, 107–113. 5. Rebbapragada,I. and Lykke-Andersen,J. (2009) Execution of ACKNOWLEDGEMENTS nonsense-mediated mRNA decay: what defines a substrate? The authors thank Prof. Jesus Jimenez-Barbero (CIB, Curr. Opin. Cell Biol., 21, 394–402. 6. Ivanov,P.V., Gehring,N.H., Kunz,J.B., Hentze,M.W. and Madrid) for his assistance and help during the NMR ex- Kulozik,A.E. (2008) Interactions between UPF1, eRFs, PABP periments and their analysis. The authors also thank Dr and the exon junction complex suggest an integrated model for Natsuko Izumi for the plasmid construction of pcDNA5/ mammalian NMD pathways. EMBO J., 27, 736–747. NTAP-SMG-9 (2-520). 7. Shyu,A.B., Wilkinson,M.F. and van Hoof,A. (2008) Messenger RNA regulation: to translate or to degrade. EMBO J., 27, 471–481. 8. Abraham,R.T. (2004) The ATM-related kinase, hSMG-1, bridges FUNDING genome and RNA surveillance pathways. DNA Repair, 3, 919–925. Spanish Ministry of Science and Innovation 9. Yamashita,A., Kashima,I. and Ohno,S. (2005) The role of (SAF2008-00451 to O.L., SAF2008-00479 to J.T.); ‘Red SMG-1 in nonsense-mediated mRNA decay. Biochim. Biophys. Tema´ tica de Investigacio´ n Cooperativa en Ca´ ncer Acta, 1754, 305–315. (RTICC)’ from the ‘Instituto de Salud Carlos III’ 10. Yamashita,A., Ohnishi,T., Kashima,I., Taya,Y. and Ohno,S. (RD06/0020/1001 to O.L. and RD06/0020/0011 to J.T.); (2001) Human SMG-1, a novel phosphatidylinositol 358 Nucleic Acids Research, 2011, Vol. 39, No. 1 3-kinase-related protein kinase, associates with components of the 17. Masse,I., Molin,L., Mouchiroud,L., Vanhems,P., Palladino,F., mRNA surveillance complex and is involved in the regulation of Billaud,M. and Solari,F. (2008) A novel role for the SMG-1 nonsense-mediated mRNA decay. Genes Dev., 15, 2215–2228. kinase in lifespan and oxidative stress resistance in Caenorhabditis 11. Kashima,I., Yamashita,A., Izumi,N., Kataoka,N., Morishita,R., elegans. PLoS One, 3, e3354. Hoshino,S., Ohno,M., Dreyfuss,G. and Ohno,S. (2006) Binding of 18. Oliveira,V., Romanow,W.J., Geisen,C., Otterness,D.M., a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon Mercurio,F., Wang,H.G., Dalton,W.S. and Abraham,R.T. (2008) junction complex triggers Upf1 phosphorylation and A protective role for the human SMG-1 kinase against tumor nonsense-mediated mRNA decay. Genes Dev., 20, 355–367. necrosis factor-alpha-induced apoptosis. J. Biol. Chem., 283, 12. Yamashita,A., Izumi,N., Kashima,I., Ohnishi,T., Saari,B., 13174–13184. Katsuhata,Y., Muramatsu,R., Morita,T., Iwamatsu,A., Hachiya,T. 19. Fuxreiter,M., Tompa,P., Simon,I., Uversky,V.N., Hansen,J.C. and et al. (2009) SMG-8 and SMG-9, two novel subunits of the Asturias,F.J. (2008) Malleable machines take shape in eukaryotic SMG-1 complex, regulate remodeling of the mRNA surveillance transcriptional regulation. Nat. Chem. Biol., 4, 728–737. complex during nonsense-mediated mRNA decay. Genes Dev., 23, 20. Tompa,P., Fuxreiter,M., Oldfield,C.J., Simon,I., Dunker,A.K. and Uversky,V.N. (2009) Close encounters of the third kind: 1091–1105. 13. Izumi,N., Yamashita,A., Iwamatsu,A., Kurata,R., Nakamura,H., disordered domains and the interactions of proteins. Bioessays, Saari,B., Hirano,H., Anderson,P. and Ohno,S. (2010) AAA+ 31, 328–335. proteins RUVBL1 and RUVBL2 coordinate PIKK activity and 21. Li,X., Romero,P., Rani,M., Dunker,A.K. and Obradovic,Z. function in nonsense-mediated mRNA decay. Sci. Signal., 3, ra27. (1999) Predicting protein disorder for N-, C-, and internal 14. Brumbaugh,K.M., Otterness,D.M., Geisen,C., Oliveira,V., regions. Genome Inform. Ser. Workshop Genome Inform., 10, Brognard,J., Li,X., Lejeune,F., Tibbetts,R.S., Maquat,L.E. and 30–40. 22. Wullschleger,S., Loewith,R., Oppliger,W. and Hall,M.N. (2005) Abraham,R.T. (2004) The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian Molecular organization of target of rapamycin complex 2. cells. Mol. Cell, 14, 585–598. J. Biol. Chem., 280, 30697–30704. 15. Azzalin,C.M., Reichenbach,P., Khoriauli,L., Giulotto,E. and 23. Clerici,M., Mourao,A., Gutsche,I., Gehring,N.H., Hentze,M.W., Lingner,J. (2007) Telomeric repeat containing RNA and RNA Kulozik,A., Kadlec,J., Sattler,M. and Cusack,S. (2009) Unusual surveillance factors at mammalian chromosome ends. Science, bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2. EMBO J., 28, 2293–2306. 318, 798–801. 24. Lee,J.H. and Paull,T.T. (2007) Activation and regulation of 16. Gehen,S.C., Staversky,R.J., Bambara,R.A., Keng,P.C. and O’Reilly,M.A. (2008) hSMG-1 and ATM sequentially and ATM kinase activity in response to DNA double-strand breaks. independently regulate the G1 checkpoint during oxidative stress. Oncogene, 26, 7741–7748. Oncogene, 27, 4065–4074. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

Download PDF
12 pages

Loading next page...
 
/lp/oxford-university-press/characterization-of-smg-9-an-essential-component-of-the-nonsense-BwcGpEJFNE

References (29)

Publisher
Oxford University Press
Copyright
The Author(s) 2010. Published by Oxford University Press.
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/gkq749
pmid
20817927
Publisher site
See Article on Publisher Site

Abstract

Published online 3 September 2010 Nucleic Acids Research, 2011, Vol. 39, No. 1 347–358 doi:10.1093/nar/gkq749 Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex 1 2,3,4 1 2,4 Israel S. Ferna ´ ndez , Akio Yamashita , Ernesto Arias-Palomo , Yumi Bamba , 1 5 1 2, Ruben A. Bartolome ´ , M. Angeles Canales , Joaquı´n Teixido ´ , Shigeo Ohno * and 1, Oscar Llorca * Centro de Investigaciones Biolo ´ gicas, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Ramiro 2 3 de Maetzu 9, 28040 Madrid, Spain, Department of Molecular Biology, Department of Microbiology and Molecular Biodefense Research, Yokohama City University School of Medicine, Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan and Dpto. Quı´mica Organica I, Facultad de Ciencias Quımicas, Univ. Complutense, Av. Complutense s/n, 28040 Madrid, Spain Received February 26, 2010; Revised and Accepted August 6, 2010 ABSTRACT SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of SMG-9 is part of a protein kinase complex, SMG1C, IDRs could facilitate the transit of SMG-9 through which consists of the SMG-1 kinase, SMG-8 and several macromolecular complexes. SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and INTRODUCTION targets for degradation mRNAs harboring prema- Eukaryotic gene expression comprises a complex set of ture translation termination codons. Here, we have biochemical reactions starting with the transcription characterized SMG-9, showing that it comprises an of the genetic information and ending in the synthesis of N-terminal 180 residue intrinsically disordered proteins. Between these two events, post-transcriptional region (IDR) followed by a well-folded C-terminal modifications and remodelling are required to assemble domain. Both domains are required for SMG-1 a mature mRNA that can be translated by the ribosome, binding and the integrity of the SMG1C complex, and several surveillance mechanisms ensure the fidelity and accuracy of these processes. Nonsense-mediated whereas the C-terminus is sufficient to interact mRNA decay (NMD) is a post-transcriptional surveil- with SMG-8. In addition, we have found that lance mechanism that, in eukaryotes, recognizes and SMG-9 assembles in vivo into SMG-9:SMG-9 and, degrades mRNAs containing premature translation ter- most likely, SMG-8:SMG-9 complexes that are not mination codons (PTCs) to prevent the accumulation of constituents of SMG1C. SMG-9 self-association is potentially harmful truncated polypeptides encoding for a driven by interactions between the C-terminal truncated protein (1,2). domains and surprisingly, some SMG-9 oligomers The NMD machinery marks a PTC-containing mRNA are completely devoid of SMG-1 and SMG-8. We for degradation through a highly sophisticated sequence propose that SMG-9 has biological functions of protein-protein interactions involving different poly- beyond SMG1C, as part of distinct SMG-9- peptides (2,3). Until recently, seven conserved core containing complexes. Some of these complexes factors for NMD had been identified to be present in most metazoan, SMG-1, Upf1, Upf2, Upf3, SMG-5, may function as intermediates potentially regulating *To whom correspondence should be addressed. Tel: +34 91 837 3112 (extn 4446); Fax: +34 91 536 0432; Email: ollorca@cib.csic.es Correspondence may also be addressed to Shigeo Ohno. Tel: +81 45 787 2596; Fax: +81 45 785 4140; Email: ohnos@med.yokohama-cu.ac.jp Present address: Israel S. Ferna´ ndez, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 348 Nucleic Acids Research, 2011, Vol. 39, No. 1 SMG-6 and SMG-7. Thanks to an intense current complex and probably up-regulates the kinase activity of research effort, a picture of the players and the molecular SMG-1 since SMG-1:SMG-9 complexes show higher mechanisms involved in NMD is starting to emerge (1,2). activity than SMG-1 (12). Both SMG-8 and SMG-9 are Yet, many aspects remain obscure and several models required for mammalian NMD and the stable association have been proposed to explain the molecular mechanisms between these two proteins is an absolute requirement for by which the NMD machinery tags an mRNA for degrad- the inhibition of the NMD response until a genuine PTC is ation (4,5). An interesting debate in current literature recognized (12). upholds a vision of NMD regulated by the outcome of a In order to improve our understanding of the functions competition between stimulating and downregulating and molecular structure of the SMG1C complex, here we signals (3–6). In any case, the topic of what determines have characterized SMG-9. We present evidence showing the fate of a PTC-containing mRNA is still the subject that SMG-9 comprises two distinct domains. The of open research (3,5,7). N-terminal domain is an intrinsically disordered region One of the central players articulating the NMD (IDR) essential for the maintenance of the structural in- response is SMG-1, a large protein of roughly 430 kDa tegrity of the SMG-1:SMG-8:SMG-9 complex. IDRs are a that belongs to the phosphatidylinositol 3-kinase-related hot topic of research, as they appear to provide the protein kinase (PIKK) family of serine-threonine kinases essential specificity and malleability for the large macro- (8,9). SMG-1 is a component of mRNA surveillance molecular machines requiring multiple and variable complexes and the phosphorylation of Upf1 by SMG-1 protein–protein interactions, as those functioning in all is the single essential event in metazoans to trigger all stages of eukaryotic gene expression (19,20). Unexpec- the latter processes leading to the degradation of an tedly, several experiments in vivo reveal that, besides is mRNA (10). A complex known as SURF participation as a component of the SMG1C complex, (SMG-1:Upf1:eRF1:eRF3) and containing SMG-1, Upf1 SMG-9 can assembly as homodimers and, most likely, and the eukaryotic release factors eRF1 and eRF3 is SMG-8:SMG-9 heterodimers that could represent inter- assembled on a termination codon together with the mediates regulating the assembly of the SMG1C complex. ribosome (11–13). The ribosome:SURF complex can interact with a downstream exon–junction-complex MATERIALS AND METHODS (EJC), a protein complex deposited 20–24 nt upstream the exon–exon junction, through the Upf2 and Upf3 Prediction of ordered and disordered regions proteins, activating the kinase activity of SMG-1 on We analysed the predicted ordered and disordered regions Upf1. Phospho-Upf1 is thought to recruit the in the sequences of SMG-1, SMG-8 and SMG-9 using one mRNA-decapping as well as the RNA-degrading machin- of the most accepted predictors of naturally disordered ery that eventually degrades the mRNA containing the regions, PONDR (http://www.pondr.com) (21). The PTC (2,3). default predictor VL-XT was used. SMG-1 has been shown to play other roles besides controlling NMD. Depletion of SMG-1 in human cells 12–180 Cloning, expression and purification of NT-SMG-9 influences the response to DNA damage (8,14) and regu- lates the association of telomeric repeat-containing RNA NT-SMG-9 cDNA was subcloned between the EcoRI and at telomeres (15). SMG-1 is required for adequate regula- NcoI sites on modified N-terminal HisTag pRAT4, pRHO tion of p53 phosphorylation upon genotoxic and oxidative and pGEX-6P-2 plasmids (GE Healthcare Bio-Sciences, stress and controls cell proliferation and apoptosis Buckinghamshire, UK). The initial GST fusion construct (14,16–18). Although the molecular bases of all these was made comprising amino acids 1–180. After the obser- vation of spontaneous self-cleavage in the initial processes are unclear, many of these functional features GST-fusion construct, the site of cleavage was identified parallel those of other PIKKs, suggesting some cooper- by mass spectroscopy and the construct re-cloned ation among the members of this family of kinases (8,13). comprising amino acids 12–180. All proteins were ex- Very recently, two novel components of a SMG-1 pressed in Escherichia coli strain BL21(DE3) and the ex- complex have been discovered and named SMG-8 and SMG-9 (12). These proteins were isolated due to their pression detected in a soluble fraction after cell lysis by co-purification with SMG-1, with which they form a sonication. GST-fusion proteins were puriEed with stable complex (SMG-1:SMG-8:SMG-9), named GST-Trap columns (20 ml, GE Healthcare Bio-Sciences) SMG1C. SMG-8 and SMG-9 are tightly associated with and NT-SMG-9 was isolated from GST after digestion SMG-1 and they seem to regulate its kinase activity and with the 3C protease. A second GST-trap column the remodelling of the mRNA surveillance complex. followed by a cation exchange chromatography in Interestingly, additional NMD factors, Ruvbl1, Ruvbl2, SP-sepharose HiTrap colum (5 ml, GE Healthcare RPB5 and SMG-10, have just been described, highlighting Bio-Sciences) followed by a final step of gel-Eltration the complexity of the NMD machinery (13). SMG-8 is a chromatography (Sephacryl S-100, GE Healthcare 991 amino acid protein which has been proposed to Bio-Sciences) yielded a highly pure preparation as regulate the correct localization of SMG-1 at the judged by SDS–PAGE after Coomassie brilliant blue PTC-stalled ribosome to form the SURF complex (12). staining. Protein concentration was determined by UV SMG-9 is a 520 amino acid protein comprising a central absorption at 280 nm. The protein solutions were putative nucleotide-triphosphatase domain. SMG-9 seems concentrated with an Amicon Ultra device (Millipore, to regulate the formation of the SMG-1:SMG-8:SMG-9 Bedford, MA, USA). Mass spectroscopy was used to Nucleic Acids Research, 2011, Vol. 39, No. 1 349 assess the identity as well as the purity of final The soluble fractions were pre-cleared with sepharose 4B preparations. (Sigma) and then incubated with streptavidin–sepharose (GE Biotech) for 2 h at 4 C with gentle rotation. Spectroscopic techniques Pre-cleared lysates were incubated with streptavidin– sepharose or anti-V5 antibodies for 2 h or 1 h at 4 C Circular dichroism (CD) spectra were recorded on a with gentle rotation. For antibodies, subsequently, the JASCO J-805 spectropolarimeter. An optical cuvette soluble fractions were incubated with 30 ml of protein G with a 1-mm path length was used. The temperature of sepharose (GE Biotech) for an additional 1 h at 4 C with the measuring cell was maintained at 25 C. Spectra were gentle rotation. After washing with RNase() T-lysis collected in a spectral range of 200–250 nm with a buffer, the affinity-purified protein complexes were path-length of 1 nm. The NT-SMG-9 preparation was eluted by incubation at 4 C for 30 min with RNase() dissolved in 20 mM sodium phosphate (pH 7.2) and lysis buffer containing 2 mM biotin (Sigma) or SDS 50 mM NaCl at a concentration of 15 mM. Data was sample buffer, respectively. All proteins in western blot analysed using the software KD2. experiments were detected with an ECL western blot Fluorescence spectra were acquired on an F-4500 detection kit (GE Biotech) or Lumi-Light (Roche). All Fuorescence spectrophotometer (Hitachi, Tokyo, Japan) experiments were performed two to three times, and at 25 C. The concentration of NT-SMG-9 was 15 mM. typical results are shown. Buffer solution contained 20 mM sodium phosphate (pH 7.2) and 50 mM NaCl. The excitation wavelength Size exclusion chromatography of SMG-9 complexes was 295 nm, and the emission spectra were recorded between 285 and 500 nm. Denaturing conditions For the preparation of HeLa cell extracts for gel column involved the measurement with the same conditions fractionation, 3  10 HeLa cells were re-suspended in an buffer and protein concentration but in the presence of equal volume of lysis buffer containing 10 mM Tris–HCl 6 M guanidinium hydrochloride (Pierce). at pH 8.0, 150 mM NaCl, 0.4% NP40, 2 mM MgCl , In the NMR spectroscopy experiments, samples for H 0.2 mM DTT, 0.1 mM PMSF, 100 nM Okadaic acid and monodimensional spectra were prepared in 20 mM sodium 200 mg/ml RNaseA. After incubation on ice for 10 min, phosphate (pH 7.2) and 50 mM NaCl at a concentration cells were lysed by 15 hand-strokes of a loose-fit of 100 mM. N-labelled samples were prepared in M9 Potter-Elvehjem homogenizer. The cell lysate was medium at a concentration of 200 mM. The NMR centrifuged at 15 000g for 30 min, and the supernatant samples contained 10% D O.The monodimensional as was loaded onto a 24 ml Superose 6 FPLC column (GE 1 15 well as H– N heteronuclear single-quantum correlation Biotech) equilibrated with lysis buffer. Fractions (400 ml) (HSQC) spectra were acquired at 25 C on a Bruker were collected from 5 to 24 ml elution volume. Fractions DMX-600 spectrometer equipped with a cryoprobe. were pooled and concentrated. Proteins were detected by western blotting. A control molecular marker was For reagents (antibodies) obtained by running the proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), Anti-SMG-8 and -SMG-1 have been described earlier aldolase (158 kDa), and RNaseA (15 kDa) (GE Biotech) [Yamashita et al. (12)]. Anti-HA (clone 3F10) (Roche), on the same column under the same conditions. anti-SBP (SantaCruz), anti-mTOR (Cell Signaling Technology), anti-aPKC (C-20) (SantaCruz) were obtained commercially. RESULTS Affinity purification, immunoprecipitation and SMG-9 comprises two distinct structural domains western blot analysis The first description of SMG-9 reported the presence of a pEF_Flag-HA-SBP-SMG-9 (2–520) (for Figure 5B), putative nucleotide-triphosphatase domain comprising pEF_Flag-HA-SBP-NT-SMG-9 (2–181), pEF_Flag- residues 181–520 (12). We now performed a more HA-SBP-CT1-SMG-9 (185–520), pEF_Flag-HA-SBP- thorough analysis of the SMG-9 sequence using several CT2-SMG-9 (175–520), pcDNA5/NTAP(CBP-SBP)- bioinformatic methods, which revealed the presence of SMG-9 (2–520) (for Figure 5C), pSR_Strep-HA-SMG-9 an unusual N-terminal region. The N-terminal region of full (2–520), pSR-V5-SMG-9 full (2–520), pSR_V5- SMG-9 was enriched in prolines, polar and charged NT-SMG-9 (2–181) and pSR-V5-CT-SMG-9 (182–520) residues while showing a low content in the hydrophobic were constructed by cloning each cDNA fragment by residues that most frequently form the hydrophobic core standard methods. siLentGene-puro-siSMG-9UTR of conventional protein domains (Supplementary Figure (siRNA sequence targeted to the 3 -UTR of the SMG-9 S1). These features are the signature of intrinsically dis- mRNA: GGAGAGGAATGTCATGCAC) was con- ordered regions (IDRs), segments of proteins that under structed by method described in manual. native conditions do not fall into a conventional fold and A 293T cells were transfected using HEKfectin which participate in specific binding to targets in highly (Biorad), and lysed with a loose-fit Potter–Elvehjem hom- complex multi-component macromolecular machines (19). ogenizer in T-buffer [20 mM HEPES–NaOH at pH 7.5, We searched for the presence of IDRs in SMG-9 based on 50 mM NaCl, 0.05% Tween-20, 2.5 mM MgCl , 0.5 mM the distinctive signature of their sequence composition and DTT, protease inhibitor cocktail (Roche), phosphatase in- conservation of amino acids. Several in silico disordered hibitor cocktail (Roche) and 100 mg/ml RNaseA (Qiagen)]. predictors have been developed, all of them showing 350 Nucleic Acids Research, 2011, Vol. 39, No. 1 1 180 181 520 higher proportion of hydrophobic residues (Figure 1C). Also, a comparison of the ratio between hydrophobicity versus net charge in several proteins placed the NT-SMG- NT-SMG-9 CT-SMG-9 (NTPs-like) 9 domain well within the group of other known intrinsic- 1.0 ally unstructured proteins whereas the C-terminal domain showed the characteristic pattern of folded domains 0.8 (Supplementary Figure S2). 0.6 0.5 0.4 The recombinant N-terminus of SMG-9 is a 20 kDa soluble monomeric domain 0.2 To further analyse the biophysical and structural 0.0 properties of the NT-SMG-9 domain, several milligrams 0 100 200 300 400 500 of highly pure protein were required, only achievable by Residue Number recombinant production. Non-specific degradation of par- tially folded heterologous proteins expressed in BC NT-SMG-9 CT-SMG-9 Escherichia coli is a common problem, which could be potentially enhanced in the case of an IDR, due to its intrinsic disordered structure. To increase the chances of success in the production of NT-SMG-9 in E. coli, we set up three parallel strategies taking advantage of this well-established prokaryotic system. We cloned the cDNA corresponding to the first 180 residues of SMG-9 (NT-SMG-9) into three different expression plasmids con- Polar Charged Hydroph. Polar Charged Hydroph. taining either (i) a N-terminal hexahistidine tag (HisTag), Figure 1. Analysis of SMG-9 primary structure. (A) PONDR analysis (ii) a N-terminal OmpA peptide inducing the secretion of of the SMG-9 amino acid sequence. A PONDR score >0.5 predicts the expressed protein to the periplasmic space and a those amino acids belonging to a disordered region. A stretch of dis- ordered amino acids of more than 50 residues are usually considered a C-terminal HisTag and (iii) a fusion protein with a gluta- disordered domain. This analysis revealed that the sequence of SMG-9 thione synthetase transferase (GST) and a site for the 3C could be divided in two regions: an N-terminal 180 residue intrinsically protease between the GST and the target construct to disordered region and a C-terminal folded domain, encoding a putative remove the tag. We found no expression when using the nucleotide-triphosphatase (NTPase)-like domain (12). Distribution of N-terminal HisTag, whereas a small secretion of the ex- types of amino acid in NT-SMG-9 (B) and the C-terminal region of SMG-9 (C). NT-SMG-9 revealed a propensity towards polar and pressed protein was observed with the periplasmic charged amino acids while the C-terminal domain was enriched in secretion-inducing vector, albeit with a high degree of hydrophobic residues. Unstructured domains frequently exhibit a low non-specific degradation (not shown, see below and content in hydrophobic amino acids and a bias towards charged and 12–180 Figure 2A for the purification of NT-SMG-9 ). The polar residues. best results were obtained with the fusion construct con- taining a GST tag. In standard conditions, GST-NT- SMG-9 was expressed as a soluble protein, but 20–30% excellent predictive value (19,20). We used PONDR of the recovered protein from the GST column was (‘Predictor OfNatural Disordered Regions’) non-specifically proteolysed. We identified the non-specific (http://www.pondr.com/) (21) to look for potential dis- cleavage site by sequencing of the N-terminus and by ordered domains in SMG-9. SMG-9 exhibited two mass spectrometry of the spontaneous truncated clearly distinct regions in its primary structure product. We found that the first 11 amino acids of (Figure 1A). A C-terminal region comprising residues NT-SMG-9 were removed non-specifically during purifi- 181–520 was predicted to conform to a conventional cation and we therefore recloned the fragment with- well-folded domain in agreement with its description as out these residues in the GST vector to produce a a putative NTPase domain based on sequence homology more stable product. This construct, GST-NT-SMG- 12–180 (CT-SMG-9 from now on). In contrast, PONDR pre- 9 , missing the first 11 amino acids, expressed as a dicted a large highly disordered region encompassing the soluble protein and was entirely stable after perform- first 180 residues of SMG-9 (NT-SMG-9 from now on) ing the purification steps described earlier (Figure 2A). (Figure 1A). The final steps during the purification protocol Disordered regions typically exhibit a higher ratio involved a second GST affinity column to remove the between the sum of polar plus charged amino acids and GST-tag, an intermediate step of cationic exchange the hydrophobic residues than structured folded domains, and a final gel filtration chromatography (Figure 2B). since they do not form a conventional hydrophobic core. This protocol allowed the efficient production of soluble 12–180 An analysis of the distribution of different types of amino NT-SMG-9 with a yield of 3–4 mg of protein per acids in SMG-9 revealed a propensity of NT-SMG-9 liter of medium (Figure 2C). towards polar and charged amino acids (Figure 1B), We characterized the hydrodynamic behaviour of 12–180 whereas the C-terminal domain of SMG-9 showed a E. coli expressed NT-SMG-9 protein in solution to Percentage PONDR Score Order Disorder Nucleic Acids Research, 2011, Vol. 39, No. 1 351 BC 12–180 Figure 2. Expression and purification of NT-SMG-9 in E. coli.(A) SDS–PAGE of three different constructs assayed to produce a soluble fragment of NT-SMG-9. Left, N-terminal hexahistidine tag (pRHT vector); middle, periplasmic secretion vector (pRHO vector); and right, GST fusion protein (pGEX-6p-2 vector). The total cell extract right before induction (column T0), 5 h after induction with 1 mM IPTG (column T5) and the soluble fraction after sonication of the T5 sample (column S), are shown. For the periplasmic construct the supernatant (column SN) of the culture of T5 is shown, since the periplasmic space of E .coli is very leaky, over-expressed proteins can be easily localized in the supernatant of the centrifuged culture. Lastly, in those two constructs where some expression was detected, the elution from an in-batch incubation of the supernatant (periplasmic secretion construction) or the soluble fraction of T5 (GST-fusion construction) with HisTrap resin or GST–sepharose resin (column O), 12–180 are shown. The pull-down protein in the GST-NT-SMG-9 construct is labelled. (B) SDS–PAGE of a purified NT-SMG-9 after the first GST-trap column before (column NC) and after a 4 h digestion (column C) with 3C protease. (C) Final preparation after applying the purification protocol described in the text. define its state of aggregation using analytical gel filtration performed through sedimentation velocity experiments and analytical ultracentrifugation (Supplementary Figure (Supplementary Figure S3B) unambiguously showed that 12–180 12–180 S3). NT-SMG-9 eluted as a single sharp peak in size NT-SMG-9 behaved as a single species in solution, exclusion chromatography, with a retention volume of with a Svedberg coefficient of 1.1 S corresponding to a 1.72 ml, compatible with a molecular mass of 20 kDa molecular weight of 19.5 ± 0.4 kDa (Supplementary after calibration of the column (Supplementary Figure Figure S3C). These results were further confirmed by sedi- S3A). Less than 5% of the protein eluted as a large aggre- mentation equilibrium analysis performed at two different 12–180 gate in the void volume, indicating that NT-SMG-9 velocities, which notably agreed well with the previous behaved as expected for a single monomeric species in data (Supplementary Figure S3D). solution. This experiment was performed in medium-high Taking all the hydrodynamic data into account, we 12–180 ionic strength conditions (300 mM NaCl) that were conclude that the NT-SMG-9 domain did not form required to avoid interaction of the protein with the any major aggregate, the predominant species in solution column matrix. In addition, ultracentrifugation ana- being a monomer, at medium-high as well as low ionic lysis at lower ionic strength conditions (50 mM NaCl) strength conditions. 352 Nucleic Acids Research, 2011, Vol. 39, No. 1 to zero in the vicinity of 222 nm, the CD-spectra of 12–180 NT-SMG-9 showed a minimum at 205 nm and negative values of ellipticity from 235 to 200 nm (Figure 3A). This suggested the presence of certain content in secondary structure. In addition, fluorescence spectra with an excitation wavelength of 295 nm revealed a significant increase in the fluorescence emission upon de- 12–180 naturation of NT-SMG-9 using 6 M guanidinium hydrochloride compared to native conditions (Figure 3B), strongly suggesting the presence of a certain degree of secondary structure quenching the fluorescence 12–180 of the two tryptophans of NT-SMG-9 in the native protein. NMR spectroscopy was performed to definitively deter- mine the presence of an IDR at the N-terminus of SMG-9. 1 12–180 A H 1D spectrum of NT-SMG-9 showed two groups of signals, a first group of thin, well-resolved peaks (6.5–8 ppm) and a group of superposed signals accumulating between 8 and 8.5 ppm (Figure 4A). This spectrum would be compatible with an unfolded polypep- tide, being the first group of sharp peaks those signals corresponding to the flexible lateral side chains and the aromatic protons whereas the N–H backbone would appear as those signals between 8 and 8.5 ppm. We also 1 15 performed H- N-HSQC 2D experiments after labelling 12–180 15 NT-SMG-9 with N (Figure 4B), and we found that the majority of the signals attributable to the N-H backbone overlapped within a very narrow H chemical shift, ranging from 7.75 to 8.5 ppm. This is a typical spectrum for intrinsically disordered regions, where defined signals (in contrast to aggregated protein) are concentrated in a narrow range (in contrast to folded proteins). Signals of the two tryptophans present in 12–180 NT-SMG-9 were detected at 10.25, 128.89 ppm and 12–180 Figure 3. Spectroscopic analyses of NT-SMG-9 .(A) Far 1 15 12–180 10.1, 127.48 ppm, H, N chemical shifts, respectively. UV-circular dichroism spectrum of NT-SMG-9 .(B) Fluorescence 12–180 spectra of NT-SMG-9 in 50 mM phosphate buffer and 50 mM These chemical shifts are characteristic of solvent NaCl (black circles) and in the same buffer but in the presence of exposed tryptophan residues as the amino acids of a dis- 6 M Guanidinium hydrochloride (white circles). ordered protein. In addition, the HSQC spectrum showed several well-dispersed peaks typical of a folded structure, suggesting the presence of some residual structure as pre- viously suggested by CD and fluorescence spectroscopy The N-terminal domain of SMG-9 is an intrinsically data. disordered region Recent studies suggest that IDRs do not show uniform The N- and C-terminal domains of SMG-9 are required structural properties, but their structure ranges from a to maintain the integrity of the SMG1C complex fully unstructured protein (‘random coils’) to partially structured regions (‘pre-molten globule’) and more We examined the relevance of the two domains of SMG-9 ‘folded’ proteins containing some elements of secondary to maintain the integrity of the SMG-1:SMG-8:SMG-9 structure (‘molten globule’) (19,20). Proteins in the first complex in cells. For this purpose, we expressed group show no secondary structure at all as well as hydro- full-length SMG-9 and fragments comprising the 2–181 dynamic dimensions like those of coiled-coils. In contrast, N-terminal (NT-SMG-9 ) and C-terminal (CT-SMG- 185–520 pre-molten globules present a core of secondary structure, 9 ) domains as SBP (Streptavidin Binding Peptide) although less dense than that found in structured proteins. tagged fusion proteins in 293T cells. To avoid the inter- To gain insight into the structural properties of NT-SMG- ference of endogenous SMG-9, this protein was down 12–180 9 we investigated its secondary structure content regulated using RNA interference targeted to using two spectroscopic techniques, ultraviolet-circular di- 3 -untranslated region of SMG-9. The expressed proteins chroism (UV–CD) and fluorescence spectroscopy were bound to Streptavidin-beads in presence of RNaseA, (Figure 3). Whereas completely unfolded polypeptides to remove interactions mediated by RNA, eluted and the are characterized by a well-defined CD spectrum with a presence of SMG-1 and SMG-8 in the pull-downed minimum in the vicinity of 200 nm and an ellipticity close material tested by western blotting (Figure 5B). Whereas Nucleic Acids Research, 2011, Vol. 39, No. 1 353 10 86420 H ppm 10.00 9.50 9.00 8.50 8.00 7.50 7.00 H ppm 12–180 1 12–180 Figure 4. NMR spectroscopy analysis of NT-SMG-9 .(A) H monodimensional spectrum of NT-SMG-9 showing the overlapping of 1 15 15 12–180 signals in a narrow chemical shift in a range centered at 8.25 ppm. (B) H- N HSQC spectrum of N-labelled NT-SMG-9 unambiguously identified this domain as inherently unstructured due to the absence of well dispersed cross peaks. The presence of well-resolved peaks and the absence of dispersion discarded any non-specific aggregation. each product was adequately expressed, only full-length of SMG-8 whereas contributions of the N- and C-terminal SMG-9 co-purified with SMG-1 and SMG-8 in presence domains of SMG-9 are required to bind SMG-1. of RNaseA. These experiments indicated that both the N- The experiment described earlier was performed after and C-terminal domains of SMG-9 are necessary for the simultaneous co-transfection of full-length HA-tagged integrity of the SMG1C complex. To map the requirement SMG-9 and the SBP-tagged constructs, and surprisingly of SMG-9 for these interactions more precisely, in a the pull-downs revealed that the full-length and two separate set of experiments, we simultaneously tested C-terminal constructs of SMG-9 tested were interacting with full-length HA-SMG-9. Unexpectedly, CT-SMG- two C-terminal constructs comprising residues 185–520 185–520 175–520 185–520 (CT-SMG-9 ) and 175–520 (CT-SMG-9 ) 9 was forming a tight complex with full-length tagged with SBP. SBP-pull-downs confirmed that SMG-9, devoid of SMG-1 and SMG-8, in striking 185–520 CT-SMG-9 was not capable of recognizing contrast with the behaviour of full length SMG-9 that 175–520 SMG-1 or SMG-8. Interestingly, CT-SMG-9 , reproducibly pulls downs a significant amount of where a small N-terminal segment flanking the SMG-1 and SMG-8 (Figure 5C). In addition, full length C-terminal domain was incorporated, was sufficient to SBP-SMG-9 was found to interact with full length recognize SMG-8 at a similar level than full-length HA-SMG-9, indicating that the association between SMG-9 (Figure 5C), whereas the recognition of SMG-1 SMG-9 molecules takes place also in the context of the was heavily impaired. These results strongly suggested full protein (Figure 5C). Hence, the existence of putative that CT-SMG-9 is directly responsible for the recognition SMG-9 oligomers was further investigated. N ppm 354 Nucleic Acids Research, 2011, Vol. 39, No. 1 A B SBP-SMG-9 1 180181 520 SMG-9 IDR NTPs-like 2 520 SBP (SMG-9) 185 520 175 520 SMG-1 2 520 SMG-8 2 520 SMG-1 182 520 SMG-8 SBP-SMG-9 V5-SMG-9 CD SBP (SMG-9) V5 (SMG-9) SMG-1 SMG-8 HA (SMG-9) 185-520 SBP (SMG-9 ) SMG-1 185-520 SBP (SMG-9 ) SMG-8 HA (SMG-9) Figure 5. Effect of NT-SMG-9 and CT-SMG-9 truncation in SMG1C assembly. (A) Schematic structures of SMG-9 construct. (B) 293T cells were transfected with the SMG-9 plasmids shown above together with plasmid expressing the siRNA targeted to 3 -UTR of SMG-9. (C) 293T cells were transfected with the SBP-tagged-SMG-9 plasmids shown above together with the HA-tagged-SMG-9 plasmid. The cells were lysed and pull downed with the streptavidn sepharose in presence of RNaseA. Pull downed products or cell lysates (input) were then probed with the antibodies shown on 185–520 the right. (D) 293T cells were transfected with the V5-tagged-SMG-9 plasmids shown above together with the SBP-tagged-CT-SMG-9 plasmid. The cells were lysed and immunoprecipitated with anti-V5 antibodies in presence of RNaseA. Immunoprecipitated products or cell lysates (input) were then probed with the antibodies shown on the right. ‘Vector’ indicates an empty vector. SMG-9 assembles into stable oligomers with SMG-9 strongly contributes to its self-association (Figure 5D). (homo-oligomers) and SMG-8 (hetero-oligomers) that are However, we failed to detect binding between NT-SMG- not part of SMG1C 2–181 185–520 9 and CT-SMG-9 (Figure 5D) and between 2–181 182–520 2–181 V5-tagged NT-SMG-9 , or CT-SMG-9 or full V5-tagged NT-SMG-9 and SBP-tagged NT-SMG- 2–181 length SMG-9 were co-expressed with SBP-tagged 9 (data not shown). These results correlate with the 185–520 12–180 CT-SMG-9 in 293T cells (Figure 5D). Pull downs finding that recombinant NT-SMG-9 behaved as a by V5 antibody revealed a significant interaction between monomer (Supplementary Figure S3). 182–520 V5-tagged CT-SMG-9 and SBP-tagged CT-SMG- The above experiments implied that SMG-9 could 185–520 9 , indicating that the C-terminal region of SMG-9 assemble other complexes besides SMG1C and we Input SBP-pull down (RNase+) V5- HA- SBP-tagged vector 2-520 175-520 185-520 Input SBP-pull down (RNase+) Input IP:V5 antibody (RNase+) vector vector 2-181 2-520 182-520 2-181 185-520 2-520 Nucleic Acids Research, 2011, Vol. 39, No. 1 355 sought a further confirmation by partially resolving the interaction of SMG-9 with SMG-1. On the other SMG-9 complexes by size exclusion chromatography hand, SMG-9 was found to interact with SMG-8 mostly (Figure 6). HeLa cell extracts were fractionated by gel fil- through its C-terminal domain. We have purified a protein comprising the N-terminal tration and the fractions analysed by denaturing electro- domain and several biophysical approaches (gel filtration phoresis and western blotting. As controls, we used chromatography, analytical ultracentrifugation, CD, and markers of molecular weight (Figure 6A, top line), UV-spectroscopy) have confirmed unambiguously that mTOR, a PIKK member that migrates as a monomer this region behaves as a compact 20 kDa domain with (290 kDa) and as a 0.7–0.8 MDa multi-protein complex the paradigmatic characteristics of unstructured domains (22), and aPKC (78 kDa). In addition to a well-resolved as well as a limited presence of secondary structure. peak corresponding to SMG1C and comprising SMG-1, Whereas misfolded proteins usually aggregate due to the SMG-8 and SMG-9, SMG-9 was detected in two add- exposure of the hydrophobic residues that form the core itional peaks. One, composed only of SMG-9 (monomer, of folded domains, an intrinsically disordered protein is 60 kDa) clearly migrated as a homo-oligomer rather soluble even in the presence of low or no secondary struc- than a monomer, and the apparent molecular weight ture due to the unusual composition of their sequences, correlated with the dimeric species previously detected enriched in polar and charged residues (19,20). The results by pull-down assays. In addition, a second peak contain- obtained by NMR spectroscopy represent the formal ing SMG-9 migrated as a larger complex, which exactly proof that the N-terminus of SMG-9 is an IDR. The ‘sig- co-migrated with SMG-8 as 400 kDa complexes, a nature’ of the mono and bi-dimensional spectra of strong indication of an SMG-8:SMG-9 complex. These NT-SMG-9 is that typical of this group of proteins with results suggested that SMG-9 has biological functions defined signals concentrated in a narrow range (19,20). beyond SMG1C, also maybe regulating SMG1C Furthermore, the combination of NMR data and the spec- assembly by means of several SMG-9-containing troscopy studies suggests that the conformation of the sub-complexes. NT-SMG-9 domain most likely fits into the category of Accordingly, we found that interfering with SMG-9 by intrinsically unstructured proteins termed ‘pre-molten expressing NT-SMG-9 and CT-SMG-9 truncated globules’, where a limited degree of secondary structure products, affected the normal response of cells to could be localized. genotoxic stress and increased susceptibility to apoptosis There is a growing interest in the functional roles of (Supplementary Figure S4 and Supplementary Data), in intrinsically disordered regions and intrinsically dis- agreement with the role described for these complexes in ordered proteins, since these seem to play important genome stability and apoptosis (3,13). HEK-293 cells roles in cellular functions such as transcription regulation, were transfected with vectors coding for full length genome surveillance, chromatin remodeling or mRNA SMG-9, NT-SMG-9 or CT-SMG-9 fragments (see processing (19,20). Algorithms designed to detect these Supplementary Data for details) and tested for suscepti- domains in the primary structure of proteins suggests bility to cisplatin, a DNA alkylating agent known to that the number and functional relevance of IDRs cause cell apoptosis. Cis-platin treatment led to a increase with the complexity of the organism. It has dose-dependent increase in cell apoptosis that was of been estimated that 25% of the total number of proteins higher extent in cells over-expressing the N-terminal or in complex eukaryotic genomes may be totally disordered C-terminal domains of SMG-9 than in cells expressing and 50% could contain at least one disordered region. the full length protein or than in mock cells. This effect IDRs seem to be adequately suited for protein–protein was associated with reduction in pro-caspase-3 levels and interactions in large macromolecular machines involving with increased processing of PARP-1. very specific but transient interactions. These domains seem to provide high specificity sustained in a large area of contact but moderate affinities facilitating interchange DISCUSSION of partners. A recent description of the interaction The activities of the SMG1C complex, containing SMG-1, between Upf1 and the C-terminal domain of Upf2 SMG-8 and SMG-9 are essential for NMD in mammals revealed that this domain of Upf2 is intrinsically dis- (12). SMG-8 and SMG-9 regulate the kinase activity of ordered and the elements of secondary structure are only SMG-1, and SMG-8 is also required to recruit SMG-1 to co-folded upon recognition of Upf1 (23). Given the com- the mRNA surveillance complex. It has been proposed plexity of NMD and more generally of the mRNA pro- that the SMG1C complex could control NMD by inhibit- cessing machinery, intrinsically disordered regions, as the ing SMG-1-mediated Upf1 phosphorylation until a one presently described for SMG-9, could be present in PTC-containing mRNA is properly recognized (12). other components of mRNA processing pathways. Here, we show that SMG-9 comprises an N-terminal We have found that SMG-9 assembles into several domain with the characteristic features of the so-called complexes apart from SMG1C, SMG-9:SMG-9 intrinsically disordered regions (IDRs) and a well-folded complexes, and most likely also SMG-8:SMG-9 C-terminal domain (12). We demonstrate that both the complexes. The finding that SMG-9 dimers could be N-terminal IDR and the C-terminal domain of SMG-9 isolated by gel filtration (Figure 6) and that a partially 185–520 are required for the integrity of the SMG1C complex. truncated dimer (CT-SMG-9 :SMG-9) is completely Both domains are implicated in SMG-1 binding, since free of SMG-1 and SMG-8 (Figure 5C) suggests that removal of either domain disrupts, totally or partially, SMG-9 dimers are not a component of the SMG1C 356 Nucleic Acids Research, 2011, Vol. 39, No. 1 molecular weight (kDa): 669 440 232 158 15 0.5 frac.# 120 40 60 molecular weight (kDa) 669 440 232 158 SMG-1 SMG-8 SMG-9 SMG-1:SMG-8:SMG-9 SMG-8:SMG-9 SMG-9 complex complex dimer mTOR aPKCλ frac.# 25 30 35 40 45 Figure 6. Several SMG-9-containing complexes can be isolated. (A and B) Size exclusion chromatography of SMG-1, SMG-8 and SMG-9 containing complexes. Fractions were run in SDS gels and the presence of either protein tested by western blot using anti-bodies specific to each component. As molecular weight markers, commercial markers (location of their elution peaks indicated in top line), mTOR (290 kDa and 0.7 MDa) and aPKC (78 kDa) were used. complex. This finding is in striking contrast to the could function as intermediaries mediating the assembly of well-characterized behaviour of SMG-9, which pull SMG1C. One possibility could be that the self-association downs SMG-1 and SMG-8 indicating that SMG-9 is a between SMG-9 molecules through the C-terminal component of SMG1C (12). On the other hand, the de- domains, could repress the interaction with SMG-8 and/ tection of SMG-8:SMG-9 complexes indicate that an as- or SMG-1, as proposed for other signalling molecules sociation between these two proteins may also regulate the (Figure 7). For instance, ATM, a member of the PIKK interaction with SMG-1 and the assembly of SMG1C. family of kinases, is activated by a transition from an in- We propose that several SMG-9 containing complexes hibited dimer under normal conditions converting into an that do not contain SMG-1 could potentially have bio- active monomer after DNA damage, a transition requiring logical functions apart from SMG1C. Thus, SMG-1 has phosphorylation of Ser1981 (24). A possible regulatory been shown to participate in the cellular stress response event driving the transitions between the homo-oligomeric (14,16–18), and we find that the expression of truncated and SMG1C-assembled states of SMG-9 could be phos- versions of SMG-9 increased the susceptibility to apoptosis phorylation. Several conserved residues of SMG-9 located (Supplementary Figure S4). In addition, we speculate that within the N-terminal disordered and the C-terminal SMG-9:SMG-9 and, probably, SMG-8:SMG-9 complexes domains are specifically phosphorylated by SMG-1 (12) absorbance 280nM Nucleic Acids Research, 2011, Vol. 39, No. 1 357 Figure 7. Model for the assembly of SMG-9-containing complexes. (Akio Yamashita and Shigeo Ohno unpublished data). Autonomous Region of Madrid (CAM S-BIO-0214-2006 Under certain circumstances, SMG-9 would be activated, to O.L.); Human Frontiers Science Program (RGP39/ ´ ´ interacting with SMG-8 by means of its C-terminal 2008 to O.L.); ‘Consejerıa de Educaciondela domain. This SMG-8:SMG-9 complex could be the Comunidad de Madrid y Fondo Social Europeo’ (to building block to assemble SMG1C. At this stage, we E.A.P.); Japan Society for the Promotion of Science (to cannot completely rule out that SMG-9 could also be a A.Y. and S.O.); Japan Science and Technology dimer within SMG1C, although current data favours a Corporation (to A.Y. and S.O.); Ministry of Education, model with an equimolar SMG-1:SMG-8:SMG-9 Culture, Sports, Science and Technology of Japan (to complex (12). An assembly pathway of SMG1C regulated S.O.); Yokohama Foundation for Advancement of at the level of SMG-9 and SMG-8 would allow the tuning Medical Science (to A.Y.). Funding for open access of the biological functions of SMG1C with the rest of the charge: Spanish Ministry of Science and Innovation. NMD machinery to either restrain or promote the activa- Conflict of interest statement. None declared. tion of SMG-1. The N-terminal disordered domain of SMG-9 participates in the recognition of SMG-1, and it would be the characteristic malleability of IDRs an REFERENCES adequate structural property to allow the transit of SMG-9 through these distinct macromolecular complexes. 1. Conti,E. and Izaurralde,E. (2005) Nonsense-mediated mRNA SMG-9 could therefore appear as an important controller decay: molecular insights and mechanistic variations across species. Curr. Opin. Cell Biol., 17, 316–325. of SMG-1 by regulating the formation of SMG1C and 2. Isken,O. and Maquat,L.E. (2008) The multiple lives of NMD consequently the activation of its kinase activity towards factors: balancing roles in gene and genome regulation. Upf1(12). Nat. Rev. Genet, 9, 699–712. 3. Nicholson,P., Yepiskoposyan,H., Metze,S., Zamudio Orozco,R., Kleinschmidt,N. and Muhlemann,O. (2009) Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions SUPPLEMENTARY DATA beyond quality control and the double-life of NMD factors. Supplementary Data are available at NAR Online. Cell Mol Life Sci., 67, 677–700. 4. Brogna,S. and Wen,J. (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. Nat. Struct. Mol. Biol., 16, 107–113. 5. Rebbapragada,I. and Lykke-Andersen,J. (2009) Execution of ACKNOWLEDGEMENTS nonsense-mediated mRNA decay: what defines a substrate? The authors thank Prof. Jesus Jimenez-Barbero (CIB, Curr. Opin. Cell Biol., 21, 394–402. 6. Ivanov,P.V., Gehring,N.H., Kunz,J.B., Hentze,M.W. and Madrid) for his assistance and help during the NMR ex- Kulozik,A.E. (2008) Interactions between UPF1, eRFs, PABP periments and their analysis. The authors also thank Dr and the exon junction complex suggest an integrated model for Natsuko Izumi for the plasmid construction of pcDNA5/ mammalian NMD pathways. EMBO J., 27, 736–747. NTAP-SMG-9 (2-520). 7. Shyu,A.B., Wilkinson,M.F. and van Hoof,A. (2008) Messenger RNA regulation: to translate or to degrade. EMBO J., 27, 471–481. 8. Abraham,R.T. (2004) The ATM-related kinase, hSMG-1, bridges FUNDING genome and RNA surveillance pathways. DNA Repair, 3, 919–925. Spanish Ministry of Science and Innovation 9. Yamashita,A., Kashima,I. and Ohno,S. (2005) The role of (SAF2008-00451 to O.L., SAF2008-00479 to J.T.); ‘Red SMG-1 in nonsense-mediated mRNA decay. Biochim. Biophys. Tema´ tica de Investigacio´ n Cooperativa en Ca´ ncer Acta, 1754, 305–315. (RTICC)’ from the ‘Instituto de Salud Carlos III’ 10. Yamashita,A., Ohnishi,T., Kashima,I., Taya,Y. and Ohno,S. (RD06/0020/1001 to O.L. and RD06/0020/0011 to J.T.); (2001) Human SMG-1, a novel phosphatidylinositol 358 Nucleic Acids Research, 2011, Vol. 39, No. 1 3-kinase-related protein kinase, associates with components of the 17. Masse,I., Molin,L., Mouchiroud,L., Vanhems,P., Palladino,F., mRNA surveillance complex and is involved in the regulation of Billaud,M. and Solari,F. (2008) A novel role for the SMG-1 nonsense-mediated mRNA decay. Genes Dev., 15, 2215–2228. kinase in lifespan and oxidative stress resistance in Caenorhabditis 11. Kashima,I., Yamashita,A., Izumi,N., Kataoka,N., Morishita,R., elegans. PLoS One, 3, e3354. Hoshino,S., Ohno,M., Dreyfuss,G. and Ohno,S. (2006) Binding of 18. Oliveira,V., Romanow,W.J., Geisen,C., Otterness,D.M., a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon Mercurio,F., Wang,H.G., Dalton,W.S. and Abraham,R.T. (2008) junction complex triggers Upf1 phosphorylation and A protective role for the human SMG-1 kinase against tumor nonsense-mediated mRNA decay. Genes Dev., 20, 355–367. necrosis factor-alpha-induced apoptosis. J. Biol. Chem., 283, 12. Yamashita,A., Izumi,N., Kashima,I., Ohnishi,T., Saari,B., 13174–13184. Katsuhata,Y., Muramatsu,R., Morita,T., Iwamatsu,A., Hachiya,T. 19. Fuxreiter,M., Tompa,P., Simon,I., Uversky,V.N., Hansen,J.C. and et al. (2009) SMG-8 and SMG-9, two novel subunits of the Asturias,F.J. (2008) Malleable machines take shape in eukaryotic SMG-1 complex, regulate remodeling of the mRNA surveillance transcriptional regulation. Nat. Chem. Biol., 4, 728–737. complex during nonsense-mediated mRNA decay. Genes Dev., 23, 20. Tompa,P., Fuxreiter,M., Oldfield,C.J., Simon,I., Dunker,A.K. and Uversky,V.N. (2009) Close encounters of the third kind: 1091–1105. 13. Izumi,N., Yamashita,A., Iwamatsu,A., Kurata,R., Nakamura,H., disordered domains and the interactions of proteins. Bioessays, Saari,B., Hirano,H., Anderson,P. and Ohno,S. (2010) AAA+ 31, 328–335. proteins RUVBL1 and RUVBL2 coordinate PIKK activity and 21. Li,X., Romero,P., Rani,M., Dunker,A.K. and Obradovic,Z. function in nonsense-mediated mRNA decay. Sci. Signal., 3, ra27. (1999) Predicting protein disorder for N-, C-, and internal 14. Brumbaugh,K.M., Otterness,D.M., Geisen,C., Oliveira,V., regions. Genome Inform. Ser. Workshop Genome Inform., 10, Brognard,J., Li,X., Lejeune,F., Tibbetts,R.S., Maquat,L.E. and 30–40. 22. Wullschleger,S., Loewith,R., Oppliger,W. and Hall,M.N. (2005) Abraham,R.T. (2004) The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian Molecular organization of target of rapamycin complex 2. cells. Mol. Cell, 14, 585–598. J. Biol. Chem., 280, 30697–30704. 15. Azzalin,C.M., Reichenbach,P., Khoriauli,L., Giulotto,E. and 23. Clerici,M., Mourao,A., Gutsche,I., Gehring,N.H., Hentze,M.W., Lingner,J. (2007) Telomeric repeat containing RNA and RNA Kulozik,A., Kadlec,J., Sattler,M. and Cusack,S. (2009) Unusual surveillance factors at mammalian chromosome ends. Science, bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2. EMBO J., 28, 2293–2306. 318, 798–801. 24. Lee,J.H. and Paull,T.T. (2007) Activation and regulation of 16. Gehen,S.C., Staversky,R.J., Bambara,R.A., Keng,P.C. and O’Reilly,M.A. (2008) hSMG-1 and ATM sequentially and ATM kinase activity in response to DNA double-strand breaks. independently regulate the G1 checkpoint during oxidative stress. Oncogene, 26, 7741–7748. Oncogene, 27, 4065–4074.

Journal

Nucleic Acids Research – Oxford University Press

Published: Jan 3, 2011

There are no references for this article.