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Published online 4 July 2008 Nucleic Acids Research, 2008, Vol. 36, No. 13 4443–4453 doi:10.1093/nar/gkn391 The structure of the 5’-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation 1 1 1 Luchezar Karagyozov , Rinesh Godfrey , Sylvia-Annette Bo¨ hmer , 1 1 2 1, Astrid Petermann , Sebastian Ho¨ lters , Arne Ostman and Frank-D. Bo¨ hmer * Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich-Schiller-University Jena, Jena, Germany and Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden Received March 14, 2008; Revised May 30, 2008; Accepted June 4, 2008 frames (uORFs). Recent genome wide analyses have ABSTRACT revealed that uAUGs and uORFs are quite common Analysis of the human protein-tyrosine phosphatase (2,3). Generally the translation of these mRNAs follows (PTP) PTPRJ mRNA detected three in-frame AUGs at the standard route for eukaryotes. The 43S scanning com- the 5’-end (starting at nt +14, +191 and +356) with no plex, composed of the 40S ribosomal subunit, Met-tRNA intervening stop codons. This tandem AUG arrange- and translation initiation factors, is attached to the m G ment is conserved between humans and the mouse cap at the 5 -end of the mRNA. Unwinding the regions and is unique among the genes of the classical PTPs. with secondary structure, the scanning proceeds towards Until now it was assumed that the principal open the 3 -end, and when an AUG triplet in a favorable con- text is encountered, the 60S ribosomal subunit is recruited reading frame (ORF) starts at AUG . Our experi- and translation initiates. The presence of an uORF ments showed that: (i) translation of the mRNA impairs the translation of the principal reading frame as synthesized under the PTPRJ promoter starts pre- the ribosomes need to reinitiate at the downstream AUG. dominantly at AUG , leading to the generation of Alternatively, the sequence environment of the upstream a 55 amino acid sequence preceding the signal pep- AUG (uAUG) may diverge from the one which is optimal tide; (ii) the longer form is being likewise correctly for recognition by the scanning complex [A/G]CCaugG processed into mature PTPRJ; (iii) the translation of (4). In this case, some of the 40S subunits will start trans- the region between AUG and AUG inhibits the 191 356 lation at the uAUG, while others will continue scanning overall expression, a feature which depends on the (‘leaky scanning’). sequence of the encoded peptide. Specifically, a It is generally assumed that the role of the uORF is to sequence of 13 amino acids containing multiple argi- secure low levels of expression of proteins which are harm- nine residues (RRTGWRRRRRRRR) confers the inhi- ful to the cell when abundant (5–7). In addition, regula- bition. In the absence of uORF these previously tory functions of uORFs have also been identified, for unrecognized characteristics of the 5’-end of the example for the CAAT enhancer binding proteins alpha and beta and for the SCL transcription factor (8,9). mRNA present a novel mechanism to suppress, The receptor-like protein-tyrosine phosphatase J and potentially to regulate translation. (PTPRJ, also designated DEP-1, CD148), a candidate tumor suppressor protein with potent anti-proliferative and anti-migratory activity, is differently expressed in dif- INTRODUCTION ferent cell types and at different cell densities (10,11). By It has been noted that a wide variety of proteins, including dephosphorylating yet only partially characterized cellular some protein kinases, growth factors, oncogenes, recep- substrates it can interfere with signal transduction down- tors and transcription factors are expressed from messen- stream of several growth factor receptors, and exerts anti- gers, which are poorly translated (1). The mRNAs of these transforming activity in cancer cell lines of different origin 0 0 proteins are characterized by a long 5 leader (5 UTR) (12–19). Therefore regulation of PTPRJ expression may with high GC content, potentially strong secondary struc- represent an important level of controlling cellular tyro- ture and the presence of short upstream open reading sine phosphorylation, and deregulation of expression may *To whom correspondence should be addressed. Tel: +49 3641 9325660; Fax: +49 3641 9325652; Email: i5frbo@rz.uni-jena.de Present address: Sebastian Ho¨ lters, Research Unit Gynecological Molecular Biology, Medical Faculty, Friedrich-Schiller-Universita¨ t Jena, Germany 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 4444 Nucleic Acids Research, 2008, Vol. 36, No. 13 contribute to carcinogenesis. Although important, the followed by firefly luciferase codons from 2 to 550 and basic mechanisms of PTPRJ expression regulation have the stop codon. In these constructs, designated as not been explored until now. (191–356)(Luc)(pcDNA3), the firefly luciferase was trans- Some of the structural features of the mRNAs discussed cribed under the strong CMV promoter and translated above are shared by the mRNA of PTPRJ, in particular a from the AUG and AUG of PTPRJ. 191 356 long, GC rich 5 leader (GC 82%). However, this region contains three AUGs, all of which are in the reading frame Constructs with frame-shift mutations and with optimized of the main protein, with no intervening stop codons. codons. Frame-shift mutations in the region between Experiments addressing the mechanisms of PTPRJ +191 and +356 (without introducing a stop codon) expression regulation showed that translation of the were performed by PCR. We inserted one A (after mRNA starts predominantly at AUG , 55 codons +200) in the codon 4 (AUG is codon 1) or deleted 191 191 upstream of the AUG , the start of the signal peptide. one C (+340) in the 5th codon preceding AUG . The 356 356 We discovered properties of the 5 leader, which have distance between the frame shifts was 139 nt, and, when hitherto not been described in other genes. In the both frame-shift mutations are present in one template, tandem arrangement of the in-frame AUGs, the codons 47 codons were altered. The optimization of codons between them are poorly translated, resulting in lower for human translation and synthesis of the fragment expression. These results uncover a previously unrecog- was done by GENEART AG (Regensburg, Germany) nized mechanism of suppressing and potentially regulating by assembling synthetic oligonucleotides and PCR pro- translation, which may be relevant not only to PTPRJ. ducts, and was verified by DNA sequencing. For further details and sequences of the wild type region ATG – ATG , the double frame-shift, and the optimized MATERIALS AND METHODS codons, see the Supplementary Data and Supplementary Figure 4. Firefly luciferase reporter constructs Reporter constructs with ATG present. ‘In-frame’ (InF) Luc Constructs with deleted codons in the 3 -end of the region and ‘Out-of-frame’ (OutF) constructs. The genomic AUG –AUG . Constructs with deleted codons (vector 191 356 region of human PTPRJ containing the putative promoter pcDNA3.1) were created by site-directed mutagenesis. and the 5 leader was amplified from BAC DNA (details in They contained sequences from +171 to +270 (codons Supplementary Data and Supplementary Figure 1). The 1–26), followed by four codons of the signal peptide and fragment (1762 bp, GenBank EF219146) was cloned into codons 2–550 of the firefly luciferase. In these clones, the NheI and BglII sites of pGL3-Basic Vector (Promega, designated as (191-)(Luc)(pcDNA3), the transcription Mannheim, Germany), which lacks eukaryotic promoter is driven by the CMV promoter, and the translation by or enhancer. The construct p1.7_InF(pGL3) contained AUG (AUG is modified to CAU, encoding His). 191 356 nucleotides from 1419 to +343 of PTPRJ (+1 is the Two double frame-shift mutants were also constructed— transcription start site, NM_002843). The arrangement of one altering the sequence of codons 4 through 13, and the the ATGs in this clone was the same as in PTPRJ—all other altering the sequence of codons 4 through 26 (for the three ATGs (ATG , ATG and ATG ) were in one 14 191 Luc sequence of the wild type and the frame-shifted codons, reading frame. Clone pNar_InF(pGL3) contained see Supplementary Figure 5). sequences 323 to +343. The pNar(17)_OutF clone was the same as pNar_InF(pGL3) but with additional 17 PTPPRJ cDNA clones nucleotides in the region between +343 and ATG . Luc The human PTPRJ cDNA has been described previously The additional nucleotides changed the reading frame. (10); accession U10886. The cDNA was inserted in The clone with deleted 3 region – pNar-Nar_InF(pGL3), the EcoRI site of the expression vector pcDNA3 contained sequences from 323 to +82. For details on all (Invitrogen, Kalsruhe, Germany) and contained at the cloning steps, see Supplementary Data. C-end a HA tag (3). Mutations of the ATGs and the double frame-shift mutation were prepared by PCR and Constructs with PTPRJ fused to firefly luciferase. The verified by sequencing. construct pNar_Luc_Fused(pGL3) expressed the firefly luciferase fused to the first five N-end amino acids of Cell culture, transfection and antibodies PTPRJ (starting with AUG ). The construct was pre- pared by PCR and contained PTPRJ sequences from HEK293 and HeLa cells cells were maintained in DMEM/ 323 to +370, followed by the firefly luciferase sequences F12 1:1 medium, and HCT116 cells in McCoy’s medium, coding amino acids 2–550. All mutations of the ATGs all supplemented with 10% FBS. All transfections were were performed by PCR and the respective clones were done with PEI (polyethylenimine, Aldrich, Cat. No. sequenced. ATG was mutated to TTG (Leu); ATG 40872-7, transfection protocol in Supplementary Data). 14 191 to AGG (Arg), and ATG to ATT (Ile). Cells were lysed in buffer with 1% Triton X100, 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM Constructs with PTPRJ 5 leader and luciferase trans- EGTA, 5% glycerol, supplemented with protease inhi- cribed from the CMV promoter. The constructs in bitors. Immunoprecipitation was performed with goat pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany) anti-firefly luciferase polyclonal antibody from contained PTPRJ sequences from +171 to +370 Chemicon (Cat. No. AB3256, Millipore, Schwalbach, Nucleic Acids Research, 2008, Vol. 36, No. 13 4445 Germany) cross-linked to Protein G Sepharose beads with specific for firefly luciferase cDNA and Renilla luciferase dimethylpimelimidate (DMP) according to standard pro- cDNA. The level of residual contamination with plasmid cedures. To determine the synthesis of HA-tagged PTPRJ, template DNA was checked with primers specific for the HEK293 cells growing in 6-well plates were transfected beta-lactamase resistance gene (for primer sequences, see with constructs in pcDNA3, together with a plasmid Supplementary Data). encoding EYFP-tagged SHP1 (20) for control of transfec- tion efficiency. The anti-HA monoclonal antibody was from Cell Signaling Technology (cat. No 2362, Frankfurt, RESULTS Germany) and the anti-SHP1 antibody was from Santa The structural features of the first exon of PTPRJ Cruz (sc-287, Heidelberg, Germany). are highly conserved Determination of dual firefly—Renilla luciferase activity Examination of the 5 -end of PTPRJ mRNA (NM_002843) showed the presence of three AUGs To determine the expression of the firefly luciferase, which are in the reading frame of the main protein. HEK293 cells were grown in 96-well plates (flat bottom, Comparison of the first exon of the human PTPRJ and clear, Greiner Bio-One, Frickenhausen, Germany, cat. No its mouse homolog (NM_008982) revealed that they share 655098) coated with 10mg/ml poly-L-lysine (Sigma- a common pattern of organization and several remarkable Aldrich, Deisenhofen, Germany). Transfection (0.2mg similarities (Figure 1A and B). First, the length of both DNA per well) was with firefly luciferase constructs exons is almost the same (451 nt human, mouse 442 nt); (or vector pGL3-Basic alone). To determine the efficiency they code for the 5 leader of the mRNA, and for 32 amino of transfection and to normalize values plasmid DNA acids of the predicted signal peptide. Second, both exons coding for Renilla luciferase was added (pRL-TK in contain three tandem AUGs in one reading frame, with no ratio 1:16 with constructs in pGL3 or pRL-CMV in intervening stop codons. The relative position and the ratio 1:40 with constructs in pcDNA3.1. Both Renilla context of the AUGs are also conserved (Table 1). expressing plasmids are from Promega, Mannheim, Third, in case translation starts at AUG (+14 to Germany). After 24 h the cells were washed with PBS, +16), or at AUG (+191 to +193), the conservation lysed with Passive Lysis Buffer (Promega, Mannheim, of the amino acid sequence is also high (77% identity, Germany) and the dual-luciferase activity was measured Figure 1C). The high degree of conservation in the (21). The expression of constructs was measured in at least PTPRJ 5 leader, notably the conservation of the position three independent experiments (8–12 wells in each experi- of AUGs, their context and the encoded amino acids sug- ment). The values were normalized as a ratio of firefly to gested that this arrangement might be of functional Renilla luciferase activity. Where indicated the fold importance (for conservation at nucleotide sequence increase of construct activity over the activity of the pro- level, see Supplementary Figure 2). moter-less pGL3 is presented. Translation of the mRNA synthesized from the PTPRJ Determination of RNA levels by Northern hybridization promoter may start at AUG as well as at AUG and real-time RT–PCR 356 191 We cloned the genomic region of the human PTPRJ (1762 HEK293 cells were transfected with constructs with bp), spanning the transcription start site into the firefly PTPRJ 5 leader and firefly luciferase transcribed from luciferase reporter vector pGL3 (see Materials and the CMV promoter (vector pcDNA3.1) together with Methods section). The reporter activity analysis of several pRL-CMV DNA (Promega, Mannheim, Germany) for 5 deletion constructs showed that the PTPRJ core pro- normalization. Total RNA was isolated with the moter is located within about 300 nucleotides upstream of RNeasy Mini kit (Qiagen, Hilden, Germany), fractionated the transcription start (Supplementary Figure 3). on 1% agarose-formaldehyde gels, blotted on Hybond N We went on to test the functional significance of the membranes (GE Healthcare, Freiburg, Germany) and above described conserved AUG arrangement. First we hybridized to DIG-labelled PCR fragment in 20% SDS, tested the possibility of translation starting upstream of 0.25 M sodium phosphate, 50% formamide buffer at 428 AUG (+356 to +358), the codon, which in the public (22). For labelling details, see Supplementary Data. database is marked as the translation start. For this we Detection of the hybrids on the blots was with anti-DIG compared the activity of various constructs with different POD Fab fragments (Roche, Penzberg, Germany) accord- arrangement of ATG , ATG and ATG (the ATG of ing to the manufacturers instructions. 14 191 Luc the firefly luciferase). The results showed invariably that For real-time RT–PCR, the RNA preparations were additionally treated with Amplification Grade DNase I the reporter activity was strongly reduced when the (Invitrogen, Karlsruhe, Germany) according to the proto- upstream AUGs (AUG and AUG ) were not in-frame 14 191 col of the manufacturer. The inactivation of the DNase I with AUG (Figure 2A). This demonstrated that initia- Luc was checked on supercoiled plasmid DNA. cDNA was tion of translation takes place in the 5 leader, at AUG prepared by the SuperScript First-Strand Synthesis (or at AUG ). This translation then proceeds not in the System for RT–PCR with oligo(dT) (Invitrogen, reading frame of the luciferase, thus preventing its expres- 12–18 Karlsruhe, Germany). Amplification of the target sion. The residual reporter activity is presumably due to cDNA was performed with the QuantiTect SYBR Green leaky scanning directed towards the ATG , as both Luc PCR Kit (Qiagen, Hilden, Germany) using primers AUG and AUG are not in a perfect context (Table 1). 14 191 4446 Nucleic Acids Research, 2008, Vol. 36, No. 13 Nar I Not I Rsr II NgoMIV Human PTPRJ 100 300 400 ATG ATG ATG 14 191 356 ORF-1 ORF-2 ORF-3 Nar I Not I Rsr II Mouse Ptprj 100 300 400 ATG ATG ATG 11 191 347 ORF-1 ORF-2 ORF-3 Human 1 MTRGGGSGSSRG---SRDRVAARWGWAPLAPPREAPARSGTRPPRGSRARLRRVAAAAAAAA 59 MTRGGG GSSRG SR+ A R GWAPLAPPREAPA RP R RARLRRVAAAAAAA Mouse 1 MTRGGGRGSSRGRGSRELGATRGGWAPLAP PREAPASLRPRPLRARRARLRRVAAAAAAA - 60 Human 60 MSPGKPGAGGAGTRRTGWRRRRRRRRQEAATTVPGLGRTAGPDSRVRGTFQGARG 114 MSPGKPGAGGAGTRRTGWRRRRRRRR E T PG G TAG RV GTFQGA+G Mouse 61 MSPGKPGAGGAGTRRTGWRRRRRRRRLETETRAPGFGHTAG---RVPGTFQGAQG 112 Human 115 MKPAAREARLPPRSPGLRWALPLLLLLLRLGQ 146 MKPAARE R PPRSPGLRWAL LLLLLR GQ Mouse 113 MKPAARETRTPPRSPGLRWALLPLLLLLRQGQ 144 Figure 1. Pattern of organization of the first exon of the PTPRJ gene (A) in humans (NM_002843) and (B) in the mouse (NM_008982). The open reading frames are indicated. Note that ATG (ATG ), ATG (ATG ) and ATG (ATG ) are in-frame with no intervening stop codons, 14 11 191 191 356 347 resulting in the same amino-acid sequence downstream of ATG (ATG ) when translation starts at any of them. (C) Conservation of the amino 356 347 acid sequences; from top to bottom—the amino acid sequences starting from AUG (AUG ), from AUG (ATG ), and the 32 amino acids of 14 11 191 191 the predicted signal peptide, starting from AUG (AUG ). Identical residues are in blue, ‘+’ represents conservative change. 356 347 luciferase with a size of 63 kDa was supported. AUG Table 1. Context of the AUG codons appeared to be largely inactive in these constructs, since a protein product of 76 kDa, corresponding to translation Human PTPRJ, codon Mouse PTPRJ, codon Sequence starting at AUG could not be detected. AUG AUG AGCCGC AUGA 14 11 Comparison of the amounts of protein with low and AUG AUG GCTGCC AUGT 191 191 higher molecular mass, shows prevalence of the 63 kDa AUG AUG CGGGGC AUGA 356 347 products (starting presumably at ATG ). This is in dis- Other Source Sequence Luc crepancy with results presented on Figure 2A, which indi- Luciferase, pGL3, ATG Accession U47295 GCCACC AUGG cated robust translation starting upstream of ATG .We Consensus (4) GCCRCC AUGG Luc R is A or G considered that low abundance of the form with higher molecular mass might be caused by differential immuno- precipitation and/or by high susceptibility to protease degradation of the protruding N-tail of the extended pro- Based on these observations, we tested directly the abil- tein. This seemed to be indeed the case, as further analysis ity of AUG and AUG to initiate translation. First, we 14 191 of luciferase translation products directly in cell lysates analyzed the length of the firefly luciferase synthesized by revealed (Figure 7C). the reporter constructs. The use of AUG , or AUG as 14 191 To estimate the relative ability of each AUG codon in start codons would give rise to longer translation products the PTPRJ mRNA to sustain translation, we designed a than translation starting at the AUG . HEK293 cells Luc fusion construct, containing all three 5 -end ATGs. This were transfected with different constructs, lysed after 0 0 construct contained (from 5 to 3 ) the PTPRJ core pro- 24 h, luciferase was immunoprecipitated and analyzed by moter (323 to 1), the 5 leader (+1 to +355), and the western blotting. As shown in Figure 2B, two luciferase sequence coding for the first five amino acids of the signal proteins were synthesized when the AUGs in the reporter peptide (+356 to +370). This sequence was fused to the were in-frame. The sizes of the luciferase bands corre- sequence of the firefly luciferase (codons 2–550) (see sponded to one product synthesized from AUG Luc Materials and Methods section). Mutations were intro- (63 kDa), and another synthesized from AUG duced to disrupt two AUGs at a time, leaving one AUG (69 kDa). This directly indicates that AUG initiates translation and that scanning is leaky. If AUG was intact, and the constructs were subjected to expression not in tandem with AUG only the synthesis of firefly analysis. As shown in Figure 3, translation can start at Luc Nucleic Acids Research, 2008, Vol. 36, No. 13 4447 Relative luciferase activity A Relative luciferase activity 0 5 10 15 20 25 0 5 10 15 20 25 ATG ATG ATG 14 191 356 ATG ATG ATG 14 191 Luc Luc Luc −323 ATG −323 ATG ATG ATG Luc 14 191 Luc −323 Luc ATG −323 Luc −323 ATG Luc −323 Luc −323 Translation product from AUG Figure 3. Contribution of the different AUGs to the activity of reporter constructs containing the PTPRJ promoter and the 5 leader, fused to the firefly luciferase. Activity of the fusion constructs (vector pGL3, 62 kDa core PTPRJ promoter) with point mutations in the ATGs (indicated Translation product by asterisks). Relative luciferase activity represents the ratio of firefly from AUG Luc luciferase and Renilla luciferase activity (pRL-TK) normalized to empty vector controls (pGL3-Basic). Figure 2. In PTPRJ promoter/luciferase reporter constructs translation can start at AUG as well as at AUG .(A) Inhibition of reporter 191 356 activity when AUG and AUG are not in-frame. Firefly luciferase 191 Luc PTPRJ cDNA constructs in pcDNA3. The ATGs in the reporter constructs were transiently transfected into HEK293 cells, and cDNA were point mutated, leaving either AUG or activity was measured (details in Materials and Methods section). AUG intact. The results again showed (Figure 4A) Relative luciferase activity represents the ratio of firefly luciferase and that both AUG and AUG have the capacity to sup- Renilla luciferase activity (pRL-TK) normalized to empty vector con- 191 356 trols (pGL3-Basic). The frame shift is indicated by a shifted firefly port translation. The mobility of PTPRJ synthesized from luciferase gene. (B) Synthesis of extended firefly luciferase from various AUG or from AUG is the same, showing a similar 191 356 constructs. HEK293 cells were transfected with the indicated con- degree of glycosylation. The protein bands are fairly structs. Cell extracts were subjected to immunoprecipitation with broad, and bands of lesser size are also detectable, reflect- anti-firefly luciferase antibodies (covalently coupled to beads), and sub- sequently immunoblotted with anti-firefly luciferase antibodies. Size ing the different extent of glycosylation of protein mole- marker, and the relative positions of the expected translation products cules present in the cell. Apparently the presence at are indicated. synthesis of additional 55 amino acids at the N-end does not prevent correct processing of PTPRJ, which involves any of the AUG codons, albeit with different efficiency. binding of the signal peptide to the recognition particle, Translation from AUG was the weakest, apparently due the subsequent correct translocation into the ER lumen, to its poor sequence context (Table 1), and the short dis- cleavage of the signal peptide, and glycosylation. It was tance from the m G cap. The potential of AUG or of interest to know whether the specific sequence of AUG to drive expression of reporter luciferase seems the additional amino acids at the N-end of PTPRJ may approximately equal. Our further results showed that play a role in the process of signal recognition. To test this translation starting at AUG is attenuated. The lack of we designed an expression construct with two frame-shift marked difference in expression starting from AUG or mutations (plus and minus) in the region between AUG AUG , implies that AUG is in a weaker sequence and AUG . These combined mutations change the 356 356 context; more ribosomes start at AUG , but the overall amino-acid sequence upstream of AUG , but leave translation is not higher. the reading frame for the signal peptide and the mature PTPRJ protein intact. Judging from mobility (Figure 4B), processing and protein glycosylation of PTPRJ were not Translation starting at AUG results in a correctly severely affected by this manipulation. We conclude that processed protein the additional amino acids preceding the signal peptide are PTPRJ is a receptor-like protein-tyrosine phosphatase compatible with correct processing, (mature protein 1302 amino acids, calculated molecular mass 150 kDa). It contains a single intracellular catalytic Translation starts predominantly at AUG domain, a single transmembrane domain, and a number of fibronectin type III repeats in its extracellular domain. An The results on the expression of PTPRJ from the cDNA N-end signal peptide of 35 amino acids has been predicted constructs as well as on the expression of the luciferase (10). The mature PTPRJ is heavily glycosylated, and reporters showed that translation could start at AUG , migrates as a 180–200 kDa protein. as well as at AUG . Further experiments were per- We investigated the possibility of PTPRJ synthesis from formed to estimate what fraction of scanning complexes the different AUGs in the 5 leader, employing HA-tagged would start translation from AUG . For this we again empty vector pGL3 control, pGL2, SV40 promoter −1419/+343_LUC out-of-frame −1419/+343_LUC in-frame −323/+343_LUC in-frame −323/+343_LUC in-frame/ ATG mutated 191 4448 Nucleic Acids Research, 2008, Vol. 36, No. 13 Relative luciferase activity 0 5 10 15 20 25 ATG ATG ATG 14 191 356 Luc −323 anti-HA PTPRJ ATG ATG ATG 14 191 356 Luc −323 anti-SHP1 ATG ATG ATG 14 191 356 Luc −323 Figure 5. Preferred initiation of translation at ATG . Wild-type con- struct, construct with plus frame shift mutation (insertion of a single nucleotide at codone 4; AUG is codone 1), and a construct with minus frame-shift mutation (deletion of a single nucleotide in codon PTPRJ anti-HA 51) were transiently transfected and luciferase activity was determined (vector pGL3, core PTPRJ promoter). Relative luciferase activity represents the ratio of firefly luciferase and Renilla luciferase activity (pRL-TK) normalized to empty vector controls (pGL3-Basic). anti-SHP1 Relative luciferase activity Figure 4. Expression of HA tagged PTPRJ with different 5 leader 0 5 10 15 20 25 sequences driven by the CMV promoter in pcDNA3. HEK293 cells ATG ATG ATG 14 191 Luc were transiently transfected with the indicated constructs expressing Luc PTPRJ with HA-epitope at the C-terminus. To normalize for transfec- −323 tion efficiency, co-transfection with hSHP-1 expression plasmid was performed (SHP-1 is not expressed endogenously in HEK293 cells). ATG ATG 14 Luc Cell extracts were subjected to SDS–PAGE and immunoblotting Luc using anti-HA antibodies and anti-hSHP-1 antibodies. (A) cDNA con- −323 structs with differentially inactivated ATGs were compared, as indi- cated. All lanes were on the same blot with identical exposure and ATG ATG 14 Luc image processing, but rearranged for better clarity. (B) The role of Luc the amino-acid sequence downstream of ATG was tested by altering −323 the sequence between ATG and ATG . 191 356 Figure 6. Inhibition of expression by translation initiating at AUG . introduced single nucleotide frame-shift mutations (one Firefly luciferase reporters (vector pGL3, core PTPRJ promoter) with insertion or one deletion) positioned in the region between non-mutated sequence, with deletion downstream of +84 and with AUG and AUG . These frame-shift mutation deviate point mutated AUG (indicated by an asterisk) were transiently trans- 191 356 fected and activity was compared. Relative luciferase activity represents the translation starting at AUG into a reading frame, the ratio of firefly luciferase and Renilla luciferase activity (pRL-TK) different from that of the luciferase. In this case, it is normalized to empty vector controls (pGL3-Basic). reasonable to assume that luciferase can be produced only by initiation of translation at AUG (resulting we prepared a deletion mutant missing nucleotides from from leaky scanning at AUG ). Both frame-shift muta- +83 to +343, eliminating part of the structured 5 leader tions caused a severe reduction of reporter activity, indi- as well as the conserved ATG . The results showed that cating that more than two-third of the scanning complexes the reporter activity of this construct is increased start translation at AUG (Figure 5). The prevalence of (Figure 6). We considered the possibility that the effect translation from AUG was confirmed by analyzing the of the +83 to +343 deletion is solely due to loss of sec- translation products by immunoblotting of the lysates of ondary structure, making scanning easier. To test this, transfected cells (see section ‘Translation of the codons ATG was eliminated by point mutation (to AGG, between AUG and AUG slows down expression’). 191 356 coding Arg). As supposed the single nucleotide change did not affect significantly the RNA secondary structure Attenuation of PTPRJ expression by a sequence (dG = 183.10). downstream of AUG Unexpectedly, however, this mutation increased repor- The highly conserved features of the 5 -end of the PTPRJ ter activity as it was the case with the deletion (Figure 6). mRNA, and the start of translation from the AUG To explain these results we made the following assump- suggest a functional significance of this region of the mes- tion, which we tested further. Translation of the codons senger. The 5 -end of the mRNA is very GC-rich (82%) that follow AUG may not be efficient, presumably and highly structured. The predicted secondary structure leading to pausing of translating ribosomes and lowering of nucleotides +1 to +358 with overall free energy the total expression. Thereby scanning through this dG = 186.70 (23) is presented in Supplementary region may be more favorable than translation itself. Figure 6. To investigate the role of this region, In case the translation from AUG is abolished, vector control all ATGs mutated only ATG active only ATG active all ATGs active ATG active 191, 356 ATG active, 191, 356 aa sequence altered only ATG active, aa sequence altered vector control Nucleic Acids Research, 2008, Vol. 36, No. 13 4449 the scanning complexes continue unrestrained towards the control Renilla luciferase—26.3, and 26.4 for transfection downstream AUG. with the wild type and the double frame-shift mutant, respectively). Thus, the increase in activity by altering the amino-acid sequence can be attributed solely to Translation of the codons between AUG and AUG 191 356 changes in translation efficiency, and not to accompanying slows down expression changes in mRNA synthesis or stability. To investigate further the causes for the inefficient transla- One possibility for the increase in reporter activity tion downstream of AUG , and to exclude that the would be the presence of rare codons in the reading effects are promoter-specific, we generated several repor- frame, which were eliminated by the frame-shift. ters in the pcDNA3 vector. The constructs downstream of Examples of translation inhibition by the occurrence of the CMV promoter contained PTPRJ sequences from rare codons have been reported for viral genes, expressed +171 to +370, which encode ATG , the downstream in mammalian cells (24,25). Inspection of the PTPRJ region (wild type or modified), ATG , plus the codons sequence from +191 to +356 showed a marked codon of the next 4 amino acids of PTPRJ, fused to the codons of bias (see Supplementary Figure 4). Out of 55 codons, 14 the firefly luciferase for amino acids 2–550 (see Materials codons are rare (http://www.kazusa.or.jp/codon/). To test and Methods section). In these reporters the luciferase the possible relevance of rare codons for inefficient trans- mRNA is transcribed from the strong cytomegalovirus lation, we used a reporter with unchanged amino acids, promoter, however, translation is dependent on the but the nucleotides between ATG and ATG were 191 356 tandem AUG and AUG (and the region between 191 356 replaced with synthetic DNA encoding the same amino them). We compared the activity of several constructs: acids but with codons optimized for human expression the wild-type construct, a construct with one frame-shift (see Materials and Methods section, and Supplementary mutation, a construct in which the sequence of 47 amino Figure 4). At the same time the overall secondary structure acids was altered by introducing two point mutations, of of the region was left largely unchanged (for the 1–284 nt which the first one altered, and the second one restored the of the mRNA transcribed from the pcDNA3 constructs reading frame, and a construct in which these 47 amino dG = 112.83 for the wild type and dG = 104.01 for the acids were deleted (Supplementary Figure 4). As expected, mRNA with optimized codons). The results showed, how- the activity of the construct with one frame-shift mutation ever, that ‘optimizing’ the codons by replacing the rare was low, again demonstrating that translation initia- codons leads only to modest increase in reporter activity tion starts predominantly at AUG . As shown in 191 (note the similarity in reporter expression of constructs Figure 7A, the reporter activity increased 2–3-fold when 1 and 4, Figure 7A). the amino acid sequence of the region between AUG 191 Another possible reason for inefficient translation could and AUG was altered or deleted. This indicated that 356 be the properties of the nascent peptide itself. The activity translation of the codons following AUG reduces the 191 of the reporter construct encoding an altered sequence overall expression of the reporter. The similar level of of a stretch of 47 amino acids was clearly elevated expression of these two constructs (3 and 5) shows addi- (compare activity of constructs 1 and 3, Figure 7A). tionally that the elimination of the region with potentially Deletion of these amino acids also resulted in enhanced strong secondary structure has no great effect on luciferase expression (construct 5, Figure 7A). It was of translation. importance to know which region of the nascent 55 The luciferase fusion constructs under the CMV amino-acid peptide would contribute specifically to promoter were also employed to test the length of the the translation attenuation. To narrow this region, we produced luciferase proteins by immunoblotting. prepared a construct encoding only the 26 N-terminal Transient transfection with CMV promoter-driven con- amino acids of the presumably inhibitory peptide structs produced enough reporter luciferase to be detected (Supplementary Figure 5). This construct showed low by immunoblotting directly in the cell lysates (as opposed luciferase reporter activity similar to that of the construct to products driven by the core PTPRJ promoter, see containing the entire 55 amino-acid sequence (Figure 7B). section ‘The region between AUG and AUG is diffi- 191 356 This indicated that the down-modulation is a property of cult to translate’). The results presented in Figure 7C show the N-terminal part of the sequence encoded downstream that the protein with higher molecular mass, correspond- of AUG . ing to translation from AUG is preferentially produced. To define more precisely the amino-acid sequence However, after immunoprecipitation the ratio of products responsible for translation attenuation we changed the is changed, disfavoring the form with higher molecular sequence of 10 amino acids, starting with codon 4 mass. Presumably either immunoprecipitation of this and preceding the arginine stretch. This modification did form is inefficient, or during immunoprecipitation degra- not lead to significant increase in luciferase expression dation of the additional amino acids takes place (see also (Figure 7B and Supplementary Figure 5). However, Figure 2B). changing the codons from 4 through 26, again by introdu- We also checked the levels of the construct mRNAs in cing two frame-shift mutations (plus and minus) resulted the cell. As detected by Northern blotting, mRNA levels in an increase of reporter activity as it was observed with were not affected by introduction of the two point frame- the altered full-length 55 amino-acid sequence (Figure 7B, shift mutations (Figure 7D). Consistent with these data, construct 4 and Figure 7B, construct 3). real-time RT-PCR revealed no significant changes of the These results indicate that translation attenuation is mRNA levels (Mean C firefly luciferase—25.1, and 25.2, caused primarily by only 13 amino acids, which include t 4450 Nucleic Acids Research, 2008, Vol. 36, No. 13 Relative luciferase activity A C 0 20406080 100 ATG ATG 1 CMV Luc ATG ATG 191 356 2 CMV Luc Translation product ATG ATG 191 356 from AUG 3 Luc CMV + − ATG ATG 191 356 62 kDa 4 CMV Luc Translation product from AUG ATG ATG 191 356 (or AUG , control) Luc Luc 5 CMV Relative luciferase activity B 0 20 40 60 80 100 D ATG 1 CMV R Luc Luc ATG Luc 2 CMV R Firefly probe ATG Renilla probe 3 CMV CMV R Luc + − 12 1 2 RNA input (relative amount) ATG 191 0.87 0.74 0.79 0.78 Ratios Luc 4 CMV CMV Luc + − 0 0 Figure 7. Activity of reporter firefly luciferase constructs driven by the CMV promoter. (A)5 truncated sequence of the PTPRJ 5 leader containing ATG and ATG was fused to the firefly luciferase (CMV promoter from vector pcDNA3.1). In the region between ATG (codon 1) and 191 356 191 ATG (codon 56) (from top to bottom): 1, the nucleotide and the encoded amino-acid sequence was left unchanged (wild type); 2, single nucleotide at codon 51 was deleted (); 3, the amino acid sequence was altered by the indicated frame-shift mutations caused by inserting a single nucleotide at codon 4, and deleting a single nucleotide at codon 51. Note that the combined frame-shift mutations alter the amino-acid sequence of the translation product between the mutations, but the reading frame of the luciferase is maintained and luciferase can be produced. 4, A synthetic nucleotide sequence encoding the wild type amino acids, but with optimized codons was inserted between ATG and ATG ; 5, the region between codons 4 191 356 and 51 was deleted without altering the reading frame (for construct sequences, see Supplementary Figure 4). The firefly luciferase reporter activity was normalized to Renilla luciferase expression also driven by a CMV promoter. (B)5 truncated sequence of the PTPRJ leader containing ATG was fused to the firefly luciferase (ATG was modied to CAT). In the region between ATG and firefly luciferase (from top to bottom): 1, the 356 191 nucleotide and the encoded amino acid sequence was left unchanged (wild type); the position of the arginine stretch is indicated (R). 2, the leader sequence was 3 truncated after codon 26 (Arg), the reading frame was maintained; 3, the amino acid sequence of the region was altered by frame- shifts at codons 4 and 13. 4, the amino acid sequence of the region was altered by frame-shifts at codons 4 and 27 (for construct sequences, see Supplementary Figure 5). (C) Size of firefly luciferase products synthesized after transfection of HE293 cells with (191–356)Luc(pcDNA3) construct [construct 1, in (A)]. The sizes of the products in total cell lysates (TCL) before and after immunoprecipitation (IP) were compared to the size of control luciferase coded by pGL2 (Promega). (D) Reporter mRNA levels in HEK 293 cells transfected with firefly luciferase fusion constructs (vector pcDNA3.1) with PTPRJ wild type or frame-shifted leader sequence as shown in (A). Total RNA isolated after transfection was loaded in two lanes (amount of RNA differed by factor of two), separated, blotted and hybridized with a DIG-labelled firefly luciferase DNA probe. To normalize, a Renilla luciferase expression construct (CMV promoter) was cotransfected, the blot was stripped and reprobed with a DIG-labelled Renilla DNA fragment (see Materials and Methods section). Cell specific and cell-density dependent differences in a homopolymer stretch of eight arginines, encoded by expression driven by the tandem AUGs are not due codons 19–26. to differences in translation attenuation Taken together, the use of AUG results in inefficient translation of the PTPRJ mRNA. The impairment is The existence of a poorly translated region at the 5 -end of caused presumably by poorly translatable codons between mRNA, which can diminish expression, provides a possi- AUG and AUG , and more specifically by codons 14–26. bility for translation control. To test for possible 191 356 +191/+356, CMV promoter (TCL) control, pGL2 +191/+356,CMV promoter (IP) Wild type Frame shift (+/−) Nucleic Acids Research, 2008, Vol. 36, No. 13 4451 100 noted that many of the 5 UTRs of the human PTPs con- wildtype tain uORFs with yet unexplored relevance for expression +/− frame-shift altered 5′ leader regulation. However none of them contains a similar tandem AUG arrangement. Only in case of hCD45/ PTPRC an in-frame upstream AUG was detected, which was, however, only two codons apart from the proposed translation start site (for details, see Supplementary Tables 1 and 2). Nevertheless, the tandem AUG arrange- ment is unlikely to be unique to PTPRJ. A recent search of available cDNA human and mouse sequences has revealed that some of the conserved AUG codons in the 5 leader are not followed by stop codons and are in the same reading frame as the main protein (3). The relevance of such tandem arrangements has, however, to the best of HT29 HeLa HEK293 HCT116 our knowledge not yet been studied. Figure 8. Translational repression by the PTPRJ 5 leader sequence in Our experiments showed that AUG is the preferred different cell lines. The CMV-promoter driven constructs 1 (wildtype) starting codon in the context of the endogenous promoter. and 3 (+/ frame-shifted 5 leader), depicted in Figure 7A, were trans- Still, apparently not all ribosomes seem to recognize fected in the indicated cell lines and reporter activity was measured relative to a cotransfected pRL-CMV Renilla luciferase expression it (leaky scanning). The least active starting codon is construct. AUG presumably due to its proximity to the m G cap and its unfavorable context (Table 1). regulation we checked the expression of some of our AUG will direct the synthesis of the same PTPRJ reporter constructs in several cell lines, and at different mature protein of 1302 amino acids, but the N-end of cell densities. As shown in Figure 8, luciferase expression (Pro-) PTPRJ is supposed to be 55 amino acids longer in the different cell types was rather different, with low than the previously predicted precursor. This has appar- expression in HT29 cells, intermediate expression in ently no effect on the functioning of the PTPRJ as HeLa cells, and high expression in HEK293 and our results showed that the N-end extended protein is HCT116 cells. It should be emphasized that the increased glycosylated in the same way as the PTPRJ, which starts expression in some cell lines cannot be explained by differ- directly with a signal peptide of 35 amino acids ential promoter activity as the reporter firefly luciferase (Figure 2B). Apparently, in each form the N-end region and the control Renilla luciferase in these experiments of the protein is recognized by the signal recognition par- are expressed under the same CMV promoter. However, ticle, cleaved by the signal peptide peptidases, the mature the ratios of expression from wild type and mutated repor- protein being correctly glycosylated. Moreover, our results ter constructs were not altered significantly. Therefore, indicate that the exact sequence of the additional 55 amino while our data suggest differences in efficiency of initiation acids is not a precondition for the protein maturation. of translation at AUG in the different cell lines, the These results are in agreement with previous data showing translation attenuation by the sequence encoded between that the signal peptide retains its function when situated AUG and AUG can be observed in all of them, 191 356 downstream of the N-end of a protein (26). indicating the generality of this mechanism for down-reg- ulation. Since PTPRJ expression is elevated in some cell The region between AUG and AUG is difficult 191 356 lines at high culture densities, we also explored the possi- to translate bility that changes in translation efficiency would contri- The analysis of the activity of the various reporter con- bute to this phenomenon. These experiments revealed structs indicated that the region downstream of AUG is that, while reporter activity was clearly increased in cell not efficiently translated, and translation of the 55 codons cultures at high density, the attenuation of translation at the N-end seems to be a rate-limiting step in the overall from AUG is still apparent in dense cells protein expression. The alteration of 47 codons of the (Supplementary Figure 7). This finding indicates that same region by shifting and restoring the reading frame upregulation of PTPRJ expression in dense cells is not resulted in marked increase in reporter activity. Impor- caused by a release from attenuated translation. tantly, this effect on translation does not depend on the promoter. Essentially the same results were obtained when transcription was driven not by the endogenous PTPRJ DISCUSSION promoter, but by the strong cytomegalovirus promoter. PTPRJ mRNA translation starts mainly at AUG Only a modest increase in the expression was observed The GC-rich 5 region of the PTPRJ mRNA is highly when the wild-type sequence was replaced by a synthetic conserved among mammals. We were particularly fragment encoding the same amino acids, but with ‘opti- attracted by the observation that a tandem arrangement mized’ codons. This suggests that the presence of rare of in-frame AUGs present in the leader sequence in the codons is not the primary reason for the observed transla- first exon is highly conserved between the human and the tion inhibition. Moreover, our results showed that the mouse (Figure 1). Therefore, we explored the putative attenuation of translation depends more specifically on regulatory importance of this arrangement. It should be 13 amino acids at the N-end of the protein. Based on Relative luciferase activity 4452 Nucleic Acids Research, 2008, Vol. 36, No. 13 these findings we propose that the translation inhibition is analyzed cell lines. Also, we did not yet observe differences due to the sequence of the nascent peptide. Examples of in the extent of translational repression at different cell translation regulation through interaction between the densities, or in cells stimulated with a variety of growth nascent peptides and the ribosome are well known for factors or cytokines (Supplementary Figure 7, and R. prokaryotes (27). The translation inhibition exerted in Godfrey, unpublished data). Further work is required to cis- by some uORF in eukaryotes depends also on their address the possibility that expression can be modulated amino-acid sequence. In these cases, according to the by a mechanism involving the tandem AUGs and the accepted model, the nascent uORF encoded peptide inter- translational repression by the specified stretch of amino acts with the ribosome and prevents its release at the acids. Alternatively, the described inhibition of translation uORF termination codon (5,7). In the mRNA of the b may be constitutive and merely serve to achieve low levels adrenergic receptor, the uORF encodes a peptide which of PTPRJ protein, thereby permitting a more effective supposedly interacts with the messenger, and thereby inhi- expression control by other mechanisms, such as tran- bits translation (28). For mammalian genes, to the best of scriptional activation. our knowledge, observations of regulatory peptides encoded by domains within the main coding sequence SUPPLEMENTARY DATA have not yet been reported. In Arabidopsis, however, a stretch of 11–13 amino-acid residues, located 80 residues Supplementary Data are available at NAR Online. from the N-terminus of the cystathionine gamma- synthetase CGS1 gene, causes a nascent peptide-mediated ACKNOWLEDGEMENTS translation elongation arrest most likely at the step of ribosome translocation (29). This work was supported by grants to F.D.B. from the We do not know yet the exact mechanism by which the DFG SFB604 (A1), BMBF FKZ 01EA0103, DFG Bo nascent stretch of 13 amino acids encoded by the PTPRJ 1043/7-1, and the EC training network MRTN-CT-2006- 5 leader causes translational attenuation. Several mechan- 035830. We thank Dr Cornelis Calkhoven for discussion, isms are feasible. One is the interaction of the nascent and Susann Mu¨ ller and Petro Zhupanyn for generation of residues with the ribosome, interfering either with some some constructs. Funding to pay the Open Access publi- of the elongation steps (the peptidyl transfer and the trans- cation charges for this article was provided by a LOM location are candidates) or with the exit of the polypeptide grant of the Medical Faculty. from the ribosome through the exit tunnel (30–32). Conflict of interest statement. None declared. Another possibility is an elongation arrest by interaction of the nascent chain with mRNA as proposed for the peptide synthesized by the uORF of the b adrenergic REFERENCES receptor mRNA. It is relevant to note that the only gen- eral feature of the nascent peptides interacting with the 1. Kozak,M. (1991) Effects of long 5 leader sequences on initiation by eukaryotic ribosomes in vitro. Gene Expr., 1, 117–125. ribosome is that they are not acidic, some being highly 2. Crowe,M.L., Wang,X.Q. and Rothnagel,J.A. (2006) Evidence for basic (27). 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Nucleic Acids Research – Oxford University Press
Published: Aug 4, 2008
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