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Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle

Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1)... Background: The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing, and weakness in the proximal limb muscles. Why alanine- expanded PABPN1 leads to muscle-specific pathology is unknown. Given the general function of PABPN1 in RNA metabolism, intrinsic characteristics of skeletal muscle may make this tissue susceptible to the effects of mutant PABPN1. Methods: To begin to understand the muscle specificity of OPMD, we investigated the steady-state levels of PABPN1 in different tissues of humans and mice. Additionally, we analyzed the levels of PABPN1 during muscle regeneration after injury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay in skeletal muscle compared to kidney. Results: Here, we show that the steady-state levels of both PABPN1 mRNA and protein are drastically lower in mouse and human skeletal muscle, particularly those impacted in OPMD, compared to other tissues. In contrast, PABPN1 levels are increased during muscle regeneration, suggesting a greater requirement for PABPN1 function during tissue repair. Further analysis indicates that modulation of PABPN1 expression is likely due to post-transcriptional mechanisms acting at the level of mRNA stability. Conclusions: Our results demonstrate that PABPN1 steady-state levels and likely control of expression differ significantly in skeletal muscle as compared to other tissues, which could have important implications for understanding the muscle-specific nature of OPMD. Keywords: PABPN1, OPMD, Skeletal muscle, Muscular dystrophy, Polyalanine expansion Background regulation of gene expression. The best characterized RNA-binding proteins regulate all steps of RNA biogen- role of PABPN1 is in mRNA polyadenylation, where esis and play a key role in post-transcriptional regulation PABPN1 stimulates poly(A) synthesis by direct inter- of gene expression [1]. Key players among these RNA- action with the nascent mRNA poly(A) tail and the poly binding proteins are the poly(A)-binding proteins, which (A) polymerase [3,4]. In a subsequent regulatory step, modulate 3′-end formation of mRNA transcripts [2]. PABPN1 decreases poly(A) polymerase elongation activ- The nuclear poly (A)-binding protein 1 (PABPN1) is a ity following addition of 250 adenine residues, thereby ubiquitously expressed protein in eukaryotes that binds dictating the length of the poly(A) tail added to mRNA with high affinity to polyadenosine RNA [2]. PABPN1 transcripts [4]. Moreover, PABPN1 is also involved in has critical roles at multiple steps in post-transcriptional regulation of alternative cleavage and polyadenylation by suppressing weak proximal polyadenylation signals [5], which can influence both gene expression and the struc- * Correspondence: acorbe2@emory.edu; gpavlat@emory.edu Department of Biochemistry, Emory University School of Medicine, Atlanta, ture of the protein produced [6]. Finally, PABPN1 has GA, USA been implicated in the polyadenylation-dependent path- Department of Pharmacology, Emory University School of Medicine, Atlanta, way of RNA decay, which targets non-protein coding GA, USA © 2013 Apponi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Apponi et al. Skeletal Muscle 2013, 3:23 Page 2 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 genes such as the long non-coding RNAs (lncRNAs) [7]. Use Committee. Adult male C57BL/6 mice between 2 to Thus, PABPN1 modulates a number of processes that 6 months of age were used in experiments. To induce are critical for controlling gene expression. regeneration, gastrocnemius muscles of male mice were In addition to playing a key role in RNA metabolism, injected with 40 μl of 1.2% BaCl [17] and collected 2, 5 PABPN1 is of significant clinical interest as mutations in and 14 days after injury. Primary myoblasts were derived the PABPN1 gene lead to oculopharyngeal muscular dys- from the hindlimb muscles of mice and cultured to >99% trophy (OPMD) [8]. This disease is caused by a small purity as previously described [18]. Cells were maintained GCN trinucleotide expansion in the coding region of in growth media (GM: Ham’s F10, 20% fetal bovine serum, PABPN1, resulting in the expansion of a stretch of 10 ala- 5ng/ml basicfibroblastgrowthfactor(bFGF), 100U/ml nines to 12 to 17 alanines in the PABPN1 N-terminus. penicillin G, 100 mg/ml streptomycin) in a humidified 5% OPMD is a late onset, autosomal dominant disease char- CO incubator at 37°C on collagen-coated dishes. For histo- acterized primarily by progressive eyelid drooping (ptosis) logic analyses, serial 10 μm sections were collected along and difficulties in swallowing [9]. Additional weakness is the length of the muscle and stained with hematoxylin and noted in proximal limb, facial and other extraocular eosin. Images were obtained using Axiovert 200 M muscles [10-12]. Disease progression is variable between microscope with a 0.30 NA 10× or 20× Plan-Neofluar patients and complications include choking, regurgitation, objective (Carl Zeiss MicroImaging, Inc., Oberkochen, aspiration and pneumonia. The pathologic hallmark of the Germany) and camera (QImaging, Surrey, Canada) disease is the presence of nuclear aggregates of PABPN1 with OpenLab 5.5.2 (PerkinElmer, Waltham, MA). in muscle [13,14]. Given the ubiquitous expression and general function of PABPN1 in RNA metabolism [15], Immunoblot analysis how mutations of this post-transcriptional regulatory fac- Tissues were homogenized in radioimmunoprecipitation tor cause a muscle-specific disease is unclear. PABPN1 is assay (RIPA)-2 buffer (50 mM Tris-HCl pH 8.0, 150 essential for both mRNA biogenesis as well as prolifera- mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% tion and differentiation of myogenic precursor cells, SDS) with protease inhibitors (Complete Mini Tablets, suggesting a critical role in muscle regeneration and main- Roche, Pleasanton, CA). Equal amounts of total pro- tenance [16]. Skeletal muscle is highly specialized for con- tein were resolved by SDS-PAGE, transferred to nitro- traction and has unique characteristics compared to other cellulose and the desired protein was detected by tissues, such as being highly regenerative and comprised immunoblotting with appropriate antibodies and en- of multinucleated post-mitotic cells, which suggests that hanced chemiluminescence. PABPN1 levels in human intrinsic characteristics of this tissue may make it more tissues were analyzed using INSTA-blot (IMGENEX, vulnerable to the effects of mutant PABPN1 than other San Diego, CA) membranes. The primary antibodies tissues that are not affected in OPMD. and concentrations used were as follows: anti-PABPN1 To begin to understand the muscle specificity of antibody 1:5,000 [16], anti-Histone H3 1:1,000 (Abcam, OPMD, we investigated the steady-state levels of the Cambridge, MA), anti-heat shock protein 90 (HSP90) PABPN1 protein in different tissues. We find that the 1:1,000 (Santa Cruz Biotechnology, Dallas, TX) and steady-state levels of PABPN1 are drastically lower in anti-tubulin 1:5,000 (Sigma-Aldrich, St. Louis, MO). Anti- skeletal muscle compared to other tissues. Strikingly, mouse or anti-rabbit IgG 1:5,000 (Jackson ImmunoResearch, craniofacial muscles, which are affected in OPMD, show West Grove, PA) were used as secondary antibodies. the lowest levels of PABPN1. We also found that PABPN1 is upregulated during muscle repair after injury. Studies of Northern blot analysis mRNA stability indicate that regulation of PABPN1 ex- Total RNA from tissues, primary cell cultures and pression is likely due to distinct post-transcriptional fluorescence-activated cell sorting (FACS)-sorted cells mechanisms in different tissues. Taken together, our re- was isolated using TRIzol (Invitrogen, Carlsbad, CA) sults demonstrate that PABPN1 steady-state levels and according to the manufacturer’s protocol. Northern likely control of expression differ significantly in skeletal blotting was performed as described previously by muscle as compared to other tissues, which could have Ausubel et al. [19]. DNA probes were generated by important implications for understanding the muscle- polymerase chain reaction (PCR) using custom primers specific nature of OPMD. (PABPN1-F 5′-CCCAGGCAATGCTGGCCCAGTGAT CATGTCTC-3′ and PABPN1-R 5′-CTAGCCCGGCC Methods CCTGTAGATTCGACCCCGGGGC-3′,c-Myc-F 5′-GA Animals and primary muscle cell culture ACTTCACCAACAGGAACTATGACCTCG-3′ and c- Experiments involving animals were performed in ac- Myc-R 5′-GGTGTCTCCTCATGCAGCACTAGG-3′), cordance with approved guidelines and ethical approval primers from SA Biosciences, Valencia, CA (peroxisome from Emory University’s Institutional Animal Care and proliferator-activated receptor gamma coactivator 1α Apponi et al. Skeletal Muscle 2013, 3:23 Page 3 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 (PGC1α); PPM03360E, glyceraldehyde 3-phosphate de- Statistical analysis hydrogenase (GAPDH); PPM02946E) or by Ambion, Statistical analysis to determine significance between Austin, TX (QuantumRNA Classic II 18S). PCR products two groups was performed using a Student’st test. were labeled with [α- P]dCTP using a random primer One-way analysis of variance (ANOVA) was used for DNA labeling system (Invitrogen, Carlsbad, CA). comparisons between multiple groups as appropriate. All statistical analyses were performed using GraphPad Prism 5.0 for Macintosh (GraphPad Software). Differ- FACS ences were considered to be statistically significant at Mononucleated cells were enzymatically isolated from P <0.05. gastrocnemius muscles 3 days after BaCl injury and fluorescently labeled with antibodies to CD31 and CD45 Results (PE), Sca-1 (PE-Cy7), and alpha-7-integrin (AlexaFluor PABPN1 levels are lower in skeletal muscle compared to 649). Propidium iodide staining was used to gate out other tissues - - dead cells from the sort. Myoblasts (CD31 /CD45 /Sca- A better understanding of the mechanisms that underlie - + - + 1 /alpha-7-integrin /PI ) and non-myogenic cells (CD31 OPMD pathology can be obtained by analyzing the func- + + - - /CD45 /Sca-1 /alpha-7-integrin /PI ) were collected using tion of PABPN1 in skeletal muscle. To begin to identify a FACSAria II (Becton-Dickinson, Franklin Lakes, NJ). muscle-specific properties of PABPN1, we first examined Isolated cells were then processed for RNA extraction. the expression of PABPN1 across different tissues. Im- munoblot analysis revealed that PABPN1 steady-state levels vary significantly among mouse tissues, with skeletal Quantitative reverse transcription (RT)-PCR muscle displaying the lowest levels of PABPN1 (Figure 1A). cDNA synthesis from 100 ng RNA was performed using The low abundance of PABPN1 in skeletal muscle could M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). result from skewed misrepresentation of this protein mRNA levels were determined by real-time PCR using within the protein pool by the uniquely high levels of the iQ SYBR Green (Bio-Rad, Hercules, CA) and iCycler iQ Real-Time Detection System and software (Bio-Rad, Hercules, CA). The relative levels of PABPN1 were de- termined by the ΔΔCt method and normalized to the housekeeping gene HPRT1. Primers were from SA Bioscences, Valencia, CA (PABPN1: PPM25445A, HPRT1: PPM03559E). mRNA decay To analyze mRNA stability in vivo, mice were injected intraperitoneally with actinomycin D (Sigma-Aldrich, St. Louis, MO) at 2.5 μg/g and quadriceps muscles and kid- ney were collected 1, 2, 4 and 6 h later. To measure mRNA stability in primary myoblasts in vitro,5 μg/ml actinomycin D was added to the growth medium and cells were harvested 0.5, 1, 2 and 4 h later. Total RNA was extracted from tissues or cells and analyzed by north- ern blot and half-lives were determined by densitometry. 5′ and 3′ RACE In order to determine the 5′ and 3′UTRs of PABPN1 Figure 1 Nuclear poly(A) binding protein 1 (PABPN1) levels are transcripts, we used the 5′ and 3′ rapid amplification low in all skeletal muscles. Lysates prepared from different (A) of cDNA ends (RACE) system (Invitrogen, Carlsbad, mouse tissues (50 μg of total protein per lane), (B) mouse muscles CA), respectively. Total RNA from either muscle or (150 μg of total protein per lane) or (C) human tissues (20 μgof testis was used as a template according to the manu- total protein per lane) were immunoblotted with anti-PABPN1 antibody. Histone H3 and heat shock protein 90 (HSP90) were used facturer’s instructions. PCR products were cloned into as loading controls for mouse samples. Amido black staining was the pCR2.1 vector (TOPO TA cloning, Invitrogen, used as the loading control for human samples. Immunoblots are Carlsbad, CA) and sequenced by Beckman Coulter representative of at least three independent sets of tissues. Genomics, Danvers, MA. Apponi et al. Skeletal Muscle 2013, 3:23 Page 4 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 cytoplasmic proteins comprising the contractile machin- The 2.1 kb transcript, which was detected in all tissues ery in this tissue. However, relatively similar levels of both but was present at low levels in muscle (Figure 2B), uti- the nuclear protein histone H3 [20] and the cytoplasmic lizes a distal polyadenylation site 851 bp downstream of protein HSP90 [21] were observed between muscle and the stop codon (Figure 2A) [23]. The 1.4 kb represents other tissues, suggesting that the nuclear protein fraction the transcript that uses a proximal polyadenylation site is not under-represented in muscle. Furthermore, analysis 66 bp downstream of the stop codon (Figure 2A) [23]. of PABPN1 levels among different mouse muscles re- This 1.4 kb mRNA variant was the predominant tran- vealed even lower levels of this protein in the craniofacial script only in testis, but was also found in other tissues muscles (masseter, tongue and pharynx), some of which at much smaller amounts (Figure 2B). Interestingly, the are muscles primarily affected in OPMD patients [12], levels of the 1.4 kb PABPN1 transcript were very high in compared to other muscles of the body (Figure 1B). Sig- testis, which correlates with the very high levels of nificantly lower levels of PABPN1 in muscle as compared PABPN1 protein observed in this tissue (Additional file to other tissues were also observed in human samples 1: Figure S1). Furthermore, with the exception of testis, (Figure 1C), suggesting that the low levels of this pro- no significant variation in the ratio between the two tein in muscle are not species-specific findings, and mRNA isoforms was observed in the analyzed tissues. this may have physiologic implications for humans. Northern blotting also revealed a band of approximately To examine whether the expression of PABPN1 is reg- 3.6 kb that was observed in all tissues (data not shown). ulated at the protein or RNA level we performed north- This transcript was previously reported and suggested to ern blot analysis (Figure 2). This analysis revealed a be either a transcript of a related gene [22] or generated strong correlation between the low levels of PABPN1 by a distinct PABPN1 promoter [23]. We performed protein and the low abundance of PABPN1 transcript in both 5′RACE and 3′RACE from kidney, muscle and mouse skeletal muscle (Figure 2B), suggesting that con- testis but failed to identify any novel PABPN1 transcript trol of PABPN1 expression occurs at the RNA level, ei- other than the 1.4 kb and 2.1 kb mRNAs variants, ther by transcriptional or post-transcriptional means. As suggesting the 3.6 kb band might indeed represent a previously reported, PABPN1 has two major mRNA vari- transcript from a related gene. Together, our results ants, a 2.1 kb and a 1.4 kb transcript (Figure 2) [22,23]. from immunoblotting and northern blotting reveal low steady-state levels of PABPN1 mRNA and protein in skeletal muscle, which is indicative of either a decrease in PABPN1 transcription or altered mRNA stability in this tissue. PABPN1 levels are increased during muscle regeneration Adult skeletal muscle is comprised primarily of post- mitotic myofibers, however, it is a highly regenerative tis- sue that undergoes extensive repair after injury (Figure 3A) [24]. In the earliest phases of muscle regeneration, inflam- matory cells invade the tissue to remove dead tissue. Subsequently, large numbers of proliferative myoblasts derived from resident stem cells undergo differenti- ation and fusion to form new myofibers. Although PABPN1 levels are very low in adult muscle tissue, levels were significantly increased during the period of extensive cellular proliferation, differentiation and fusion that occurs 2 to 5 days after muscle injury (Figure 3B). However, 14 days after injury, when muscle architecture was restored (Figure 3A), PABPN1 levels were again low (Figure 3B). A similar pattern of upregulation was ob- Figure 2 Nuclear poly(A) binding protein 1 (PABPN1) mRNA served for the cytoplasmic poly(A) binding protein, levels are low in skeletal muscle. (A) Structure of the 2.1 kb and PABPC1 [2], during muscle regeneration, however the 1.4 kb PABPN1 transcripts (solid boxes represent coding regions and levels of the heat shock protein HSP90 remained constant open boxes non-coding regions). (B) Total RNA from different mouse tissues was analyzed by northern blot using a PABPN1 probe. over the time course. This result indicates that increased Two different exposures, short and long, are shown. 18S rRNA was levels of poly(A)-binding proteins during muscle regener- probed as a loading control. Figure is representative of at least three ation are not unique to PABPN1. To determine whether independent sets of tissues. the increased levels of PABPN1 observed at 2 to 5 days Apponi et al. Skeletal Muscle 2013, 3:23 Page 5 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 Figure 3 Nuclear poly(A) binding protein 1 (PABPN1) levels are increased during muscle regeneration in part due to increased levels in myoblasts. (A) Representative hematoxylin and eosin stained sections of gastrocnemius muscles at different times after BaCl injury are shown. (B) Lysates were prepared from gastrocnemius muscles at different times after injury and were immunoblotted with anti-PABPN1, PABPC1 or heat shock protein 90 (HSP90) antibodies (n = 3 per timepoint). (C) Total RNA was obtained from uninjured and injured muscle tissue (three independent samples) as well as fluorescence-activated cell sorting (FACS)-sorted myoblasts and non-myogenic cells obtained 3 days after muscle injury (pooled from five mice). PABPN1 mRNA levels were determined using real-time polymerase chain reaction (PCR) and hypoxanthine- guanine phosphoribosyltransferase (HPRT) mRNA was used as an internal control. Amount of PABPN1 mRNA relative to uninjured muscle is shown; n = 3. Data are mean ± SD; *P <0.05 vs uninjured muscle. after injury were due in part to myoblasts, we used flow all tissues but testis and contains a putative ARE, may be cytometry and specific antibodies to isolate myoblasts and subject to post-transcriptional regulation in different tis- non-myogenic cells (including inflammatory cells) from sues. To assess if this transcript is differently regulated in mouse muscles 3 days after injury. As we were unable to muscle, we analyzed PABPN1 mRNA stability in muscle perform immunoblots for PABPN1 on the small amount and kidney after blocking transcription in mice with acti- of cells isolated by flow cytometry, we used quantitative nomycin D. We observed that the half-life of the 2.1 kb RT-PCR to examine PABPN1 transcript levels in sorted PABPN1 transcript was significantly shorter in muscle cells compared to muscle tissue. Similar to what we ob- (2.3 h) compared to kidney (>6 h) (Figure 4A,E; served for PABPN1 protein, PABPN1 transcript levels Table 1). As a control to demonstrate similar transcrip- were increased approximately fivefold in injured com- tional inhibition between both tissues, we analyzed the pared to uninjured muscle (Figure 3C). We also found that stability of PGC1α and GAPDH mRNAs, known un- PABPN1 mRNA levels were exceptionally high in both stable and stable transcripts, respectively [25,26]. As - - - + sorted myoblasts (CD31 /CD45 /Sca-1 /alpha-7-integrin ) expected, PGC1α mRNA displayed a short half-life in + + + and non-myogenic cells (CD31 /CD45 /Sca-1 /alpha-7-in- both muscle and kidney (1.7 h and 2.6 h) compared to tegrin ) compared to uninjured muscle tissue (Figure 3C). GAPDH mRNA (>>6 h in both tissues) (Figure 4A,E; These data suggest that myoblasts significantly contribute Table 1). These results demonstrate a strong correl- to increased PABPN1 levels in regenerating muscle. We ation between the high steady-state levels of PABPN1 conclude that PABPN1 levels are not static in muscle but protein and the stable transcript in kidney, whereas in rather modulated by the physiologic state of the tissue, skeletal muscle, the low steady-state levels of PABPN1 suggesting a greater requirement for PABPN1 function dur- correlate with the unstable PABPN1 transcript. ing tissue repair. As shown earlier (Figure 3C), PABPN1 levels are mod- ulated during muscle regeneration and myoblasts con- PABPN1 mRNA is unstable in skeletal muscle tribute in part to the increased levels of PABPN1 during The two PABPN1 transcripts schematized in Figure 2A this process. We next investigated if the increase in arise from the usage of two different polyadenylation PABPN1 levels in myoblasts is accompanied by a corre- sites. The smaller 1.4 kb transcript contains virtually no sponding increase in PABPN1 mRNA stability. Similar 3′UTR, whereas the longer 2.1 kb transcript harbors a to myoblasts directly isolated from injured muscle, we 3′UTR of 851 bp containing a putative mRNA AU-rich found that cultured primary mouse myoblasts displayed destabilizing element (ARE) [23]. We hypothesized that the higher levels of PABPN1 mRNA compared to uninjured 2.1 kb transcript, which is the most abundant transcript in muscle tissue (Figure 4B). Consistent with this finding, Apponi et al. Skeletal Muscle 2013, 3:23 Page 6 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 Figure 4 Nuclear poly(A) binding protein 1 (PABPN1) mRNA is unstable in muscle tissue but stable in cultured myoblasts. (A) Total RNA was collected at different timepoints after injection of actinomycin D to inhibit transcription and PABPN1 mRNA decay was analyzed by northern blot. Time courses are shown for samples from muscle and kidney. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known unstable and stable transcript, respectively, were probed as controls (n = 3 per timepoint). To visualize PABPN1 signal in muscle samples, the blot was exposed significantly longer than for kidney samples. (B) Total RNA was obtained from skeletal muscle (SM) and cultured primary mouse myoblasts (Mb) and PABPN1 mRNA levels were determined using real-time polymerase chain reaction (PCR) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA was used as an internal control. The amount of PABPN1 mRNA relative to skeletal muscle (SM) is shown; n = 3 independent samples. Data are mean ± SD; *P <0.05 vs skeletal muscle. (C) Protein extracts were prepared from SM and Mb and immunoblotted with anti-PABPN1 antibody. HSP90 was used as a loading control. The immunoblot is representative of at least three independent samples. (D) Total RNA was collected at different timepoints after treatment of cultured primary mouse myoblasts with actinomycin D and PABPN1 mRNA decay was analyzed by northern blot; c-Myc and GAPDH, known unstable and stable transcripts in myoblasts, respectively, were probed as controls. Averages of densitometric measurements of northern blot bands were used to determine mRNA decay. The image is representative of at least three independent samples. (E) The decay profile of PABPN1 mRNA in muscle, kidney and cultured myoblasts plotted as mRNA amount relative to timepoint T = 0 h (n = 3 samples per timepoint). Data are mean ± SD. steady-state levels of PABPN1 protein were also higher in myoblasts present during muscle regeneration, is due at cultured myoblasts compared to muscle tissue (Figure 4C). least in part to the increase in PABPN1 mRNA stability in We analyzed the stability of PABPN1 transcripts in cul- those cells compared to muscle tissue. Taken together, our tured myoblasts. As observed in skeletal muscle tissue, the results suggest that PABPN1 expression in different 2.1 kb PABPN1 transcript was the predominant transcript tissues or during muscle regeneration is regulated by a in myoblasts (data not shown). However, in contrast to post-transcriptional mechanism that modulates tran- muscle tissue, the 2.1 kb PABPN1 transcript was ex- script stability. tremely stable in myoblasts (Figure 4D,E; Table 1). As expected, c-Myc mRNA, a known unstable transcript in Discussion myoblasts [27], had a short half-life compared to the Studying PABPN1 specifically in skeletal muscle is crit- much longer half-life for GAPDH mRNA, a known stable ical for defining the mechanisms which make this tissue transcript (Table 1). These results indicate that the high uniquely susceptible to the mutation causing OPMD. levels of PABPN1 in cultured myoblasts, and likely in Here, we report that steady-state levels of PABPN1 mRNA and protein are low in skeletal muscle and that Table 1 Nuclear poly(A) binding protein 1 (PABPN1) expression of PABPN1 in this tissue is controlled, at mRNA is unstable in muscle tissue but stable in cultured least in part, by post-transcriptional regulation of RNA myoblasts levels. We also demonstrate that PABPN1 levels are Muscle Kidney Myoblast modulated during muscle repair providing further sup- PABPN1 2.3 h >>6 h >>6 h port for regulation of PABPN1 expression in this tissue. PGC1α 1.7 h 2.6 h ND PABPN1 is not the only ubiquitous protein with a gen- GAPDH >>6 h >>6 h >>6 h eral function in basic cellular processes whose expression level is variable among tissues [28,29]. For example, the ex- c-Myc ND ND 0.3 h pression of histone H3A, transcription elongation factor GAPDH glyceraldehyde 3-phosphate dehydrogenase, ND not determined, PGC1α peroxisome proliferator-activated receptor gamma coactivator 1α. A1 (TCEA1) and heterogeneous nuclear ribonucleoprotein Apponi et al. Skeletal Muscle 2013, 3:23 Page 7 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 (hnRNP) C is relatively constant among different tissues, main 2.1 kb PABPN1 transcript expressed in skeletal whereas levels of GAPDH, β-actin and histone H2A are muscle harbors an ARE in the 3′UTR, we speculate this among the most variable within tissues [29]. These differ- pathway is a strong candidate for the control of PABPN1 ences in expression levels are most likely related to intrin- levels in skeletal muscle. sic properties of individual tissues and reflect differences in The specific PABPN1 expression pattern observed in metabolic activity and cellular structure. skeletal muscle may be an important feature that makes The extremely low levels of PABPN1 in skeletal this tissue more susceptible than others to the mutations muscle compared to other tissues may indicate a low re- in PABPN1 that cause the muscle-specific disease OPMD. quirement for this factor in basal muscle metabolism Whether the alanine expansion in PABPN1 leads to a and maintenance. Skeletal muscle is distinctly character- gain-of function or loss-of-function of this protein is un- ized by multinucleated, post-mitotic cells with a very known [12,15]. The nuclear aggregates observed in muscle specialized function and low complexity transcriptome of OPMD patients may exert toxic effects in the tissue as [30,31]. In skeletal muscle, a small number of genes con- hypothesized for other polyglutamine and polyalanine ex- tribute to a large fraction of the total mRNA pool, with pansion disorders [37-40]. However, as wild-type PABPN1 the ten most expressed genes in muscle accounting for can form reversible aggregates in neurons in response to 20% to 40% of the total mRNA [31]. The most abundant changes in cell physiology without overt pathology [41], transcripts in skeletal muscle encode proteins involved the toxicity of PABPN1 nuclear aggregates is unlikely to in contraction, glucose metabolism, ATP production and be the exclusive cause of OPMD etiology. In the loss-of ribosomal proteins [30,31], consistent with the role of -function model of OPMD etiology, one mechanism that this tissue in movement and metabolism. Such tran- could lead to a loss or decrease of PABPN1 function is an scripts encoding proteins involved in general cellular intrinsic reduction in PABPN1 activity caused by the ala- functions are usually stable with low turnover [27,32]. nine expansion. Although wild-type and mutant PABPN1 Therefore, the low complexity of the skeletal muscle appear to have similar polyadenylation activity in vitro [2], transcriptome associated with low turnover of a signifi- the effects of the alanine expansion on this or other cant fraction of its transcripts may explain why skeletal PABPN1 functions have not yet been addressed in the muscle has low requirements for a protein involved in context of skeletal muscle in vivo. Another mechanism mRNA metabolism such as PABPN1. that could explain a loss of PABPN1 function is the deple- Our data indicate the low levels of PABPN1 in skeletal tion of the soluble and functional fraction of PABPN1 by muscle are, at least in part, determined at the level of regulation of PABPN1 transcript stability. Regulation of mRNA decay rate is a key factor in determining the ex- pression pattern of many genes allowing rapid adapta- tion to changing cellular requirements [33,34]. PABPN1 levels increase significantly during skeletal muscle regen- eration suggesting a greater requirement for PABPN1 in myoblasts and non-myogenic cells such as inflammatory cells, which may be due to their highly proliferative sta- tus and to a more complex transcriptome compared to uninjured muscle tissue. As the increased levels of PABPN1 in regenerating muscle correlate with an increased tran- script stability in myoblasts and subsequent increase in the steady-state levels of PABPN1 transcript, we suggest that skeletal muscle employs a post-transcriptional mechanism to control PABPN1 levels according to the tissue requirements. Figure 5 Low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle may predispose this tissue to the mRNA decay rates are modulated by an interplay of deleterious effects of alanine-expanded PABPN1. We show specific stabilizing or destabilizing factors with the tran- muscle has lower levels of PABPN1 compared to other tissues in script, such as RNA-binding proteins and/or miRNAs normal individuals (N) but these levels are adequate for normal and their associated enzymes [35]. One of the most tissue function. In patients with oculopharyngeal muscular dystrophy studied post-transcriptional pathways is orchestrated by (OPMD), functional levels of PABPN1 could be decreased in all tissues due to expression of mutant PABPN1. However, muscle- a variety of RNA-binding proteins that interact with AU- specific pathology ensues in autosomal dominant OPMD because rich elements (ARE) within the 3′UTR of mRNAs the levels of PABPN1 fall below the threshold required to maintain [33,36] and many unstable mRNAs expressed in muscle proper tissue function. contain AU-rich elements in their 3′UTRs [27]. As the Apponi et al. Skeletal Muscle 2013, 3:23 Page 8 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 sequestration in the nuclear aggregates present in muscle Authors' contributions LHA, AHC and GKP conceived and designed the study. LHA performed the of OPMD patients. In fact, a recent study that examined research. LHA, AHC and GKP analyzed the research and wrote the PABPN1 transcript levels in human muscle samples manuscript. All authors read and approved the final manuscript. reported a decrease in steady-state levels of PABPN1 mRNA after the fifth decade of life, the common age for Acknowledgements We thank Matthew Randolph for assistance with flow cytometry onset of OPMD symptoms [42]. This study also presented experiments. This work was supported by the Muscular Dystrophy evidence that this decrease in PABPN1 transcript levels is Association (MDA157523, MDA68022), and by the National Institutes of accelerated in OPMD patients [42]. These recent findings Health (NS059340, AR061987). support the idea that a loss of PABPN1 function could Received: 30 May 2013 Accepted: 28 August 2013 contribute to the muscle-specific pathology in OPMD. Published: 1 October 2013 Consistent with this idea, overexpression of wild-type PABPN1 reduces the pathology caused by the expression References 1. 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Berciano MT, Villagra NT, Ojeda JL, Navascues J, Gomes A, Lafarga M, Carmo-Fonseca M: Oculopharyngeal muscular dystrophy-like nuclear • No space constraints or color figure charges inclusions are present in normal magnocellular neurosecretory neurons • Immediate publication on acceptance of the hypothalamus. Hum Mol Genet 2004, 13:829–838. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle

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Copyright © 2013 by Apponi et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing, and weakness in the proximal limb muscles. Why alanine- expanded PABPN1 leads to muscle-specific pathology is unknown. Given the general function of PABPN1 in RNA metabolism, intrinsic characteristics of skeletal muscle may make this tissue susceptible to the effects of mutant PABPN1. Methods: To begin to understand the muscle specificity of OPMD, we investigated the steady-state levels of PABPN1 in different tissues of humans and mice. Additionally, we analyzed the levels of PABPN1 during muscle regeneration after injury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay in skeletal muscle compared to kidney. Results: Here, we show that the steady-state levels of both PABPN1 mRNA and protein are drastically lower in mouse and human skeletal muscle, particularly those impacted in OPMD, compared to other tissues. In contrast, PABPN1 levels are increased during muscle regeneration, suggesting a greater requirement for PABPN1 function during tissue repair. Further analysis indicates that modulation of PABPN1 expression is likely due to post-transcriptional mechanisms acting at the level of mRNA stability. Conclusions: Our results demonstrate that PABPN1 steady-state levels and likely control of expression differ significantly in skeletal muscle as compared to other tissues, which could have important implications for understanding the muscle-specific nature of OPMD. Keywords: PABPN1, OPMD, Skeletal muscle, Muscular dystrophy, Polyalanine expansion Background regulation of gene expression. The best characterized RNA-binding proteins regulate all steps of RNA biogen- role of PABPN1 is in mRNA polyadenylation, where esis and play a key role in post-transcriptional regulation PABPN1 stimulates poly(A) synthesis by direct inter- of gene expression [1]. Key players among these RNA- action with the nascent mRNA poly(A) tail and the poly binding proteins are the poly(A)-binding proteins, which (A) polymerase [3,4]. In a subsequent regulatory step, modulate 3′-end formation of mRNA transcripts [2]. PABPN1 decreases poly(A) polymerase elongation activ- The nuclear poly (A)-binding protein 1 (PABPN1) is a ity following addition of 250 adenine residues, thereby ubiquitously expressed protein in eukaryotes that binds dictating the length of the poly(A) tail added to mRNA with high affinity to polyadenosine RNA [2]. PABPN1 transcripts [4]. Moreover, PABPN1 is also involved in has critical roles at multiple steps in post-transcriptional regulation of alternative cleavage and polyadenylation by suppressing weak proximal polyadenylation signals [5], which can influence both gene expression and the struc- * Correspondence: acorbe2@emory.edu; gpavlat@emory.edu Department of Biochemistry, Emory University School of Medicine, Atlanta, ture of the protein produced [6]. Finally, PABPN1 has GA, USA been implicated in the polyadenylation-dependent path- Department of Pharmacology, Emory University School of Medicine, Atlanta, way of RNA decay, which targets non-protein coding GA, USA © 2013 Apponi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Apponi et al. Skeletal Muscle 2013, 3:23 Page 2 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 genes such as the long non-coding RNAs (lncRNAs) [7]. Use Committee. Adult male C57BL/6 mice between 2 to Thus, PABPN1 modulates a number of processes that 6 months of age were used in experiments. To induce are critical for controlling gene expression. regeneration, gastrocnemius muscles of male mice were In addition to playing a key role in RNA metabolism, injected with 40 μl of 1.2% BaCl [17] and collected 2, 5 PABPN1 is of significant clinical interest as mutations in and 14 days after injury. Primary myoblasts were derived the PABPN1 gene lead to oculopharyngeal muscular dys- from the hindlimb muscles of mice and cultured to >99% trophy (OPMD) [8]. This disease is caused by a small purity as previously described [18]. Cells were maintained GCN trinucleotide expansion in the coding region of in growth media (GM: Ham’s F10, 20% fetal bovine serum, PABPN1, resulting in the expansion of a stretch of 10 ala- 5ng/ml basicfibroblastgrowthfactor(bFGF), 100U/ml nines to 12 to 17 alanines in the PABPN1 N-terminus. penicillin G, 100 mg/ml streptomycin) in a humidified 5% OPMD is a late onset, autosomal dominant disease char- CO incubator at 37°C on collagen-coated dishes. For histo- acterized primarily by progressive eyelid drooping (ptosis) logic analyses, serial 10 μm sections were collected along and difficulties in swallowing [9]. Additional weakness is the length of the muscle and stained with hematoxylin and noted in proximal limb, facial and other extraocular eosin. Images were obtained using Axiovert 200 M muscles [10-12]. Disease progression is variable between microscope with a 0.30 NA 10× or 20× Plan-Neofluar patients and complications include choking, regurgitation, objective (Carl Zeiss MicroImaging, Inc., Oberkochen, aspiration and pneumonia. The pathologic hallmark of the Germany) and camera (QImaging, Surrey, Canada) disease is the presence of nuclear aggregates of PABPN1 with OpenLab 5.5.2 (PerkinElmer, Waltham, MA). in muscle [13,14]. Given the ubiquitous expression and general function of PABPN1 in RNA metabolism [15], Immunoblot analysis how mutations of this post-transcriptional regulatory fac- Tissues were homogenized in radioimmunoprecipitation tor cause a muscle-specific disease is unclear. PABPN1 is assay (RIPA)-2 buffer (50 mM Tris-HCl pH 8.0, 150 essential for both mRNA biogenesis as well as prolifera- mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% tion and differentiation of myogenic precursor cells, SDS) with protease inhibitors (Complete Mini Tablets, suggesting a critical role in muscle regeneration and main- Roche, Pleasanton, CA). Equal amounts of total pro- tenance [16]. Skeletal muscle is highly specialized for con- tein were resolved by SDS-PAGE, transferred to nitro- traction and has unique characteristics compared to other cellulose and the desired protein was detected by tissues, such as being highly regenerative and comprised immunoblotting with appropriate antibodies and en- of multinucleated post-mitotic cells, which suggests that hanced chemiluminescence. PABPN1 levels in human intrinsic characteristics of this tissue may make it more tissues were analyzed using INSTA-blot (IMGENEX, vulnerable to the effects of mutant PABPN1 than other San Diego, CA) membranes. The primary antibodies tissues that are not affected in OPMD. and concentrations used were as follows: anti-PABPN1 To begin to understand the muscle specificity of antibody 1:5,000 [16], anti-Histone H3 1:1,000 (Abcam, OPMD, we investigated the steady-state levels of the Cambridge, MA), anti-heat shock protein 90 (HSP90) PABPN1 protein in different tissues. We find that the 1:1,000 (Santa Cruz Biotechnology, Dallas, TX) and steady-state levels of PABPN1 are drastically lower in anti-tubulin 1:5,000 (Sigma-Aldrich, St. Louis, MO). Anti- skeletal muscle compared to other tissues. Strikingly, mouse or anti-rabbit IgG 1:5,000 (Jackson ImmunoResearch, craniofacial muscles, which are affected in OPMD, show West Grove, PA) were used as secondary antibodies. the lowest levels of PABPN1. We also found that PABPN1 is upregulated during muscle repair after injury. Studies of Northern blot analysis mRNA stability indicate that regulation of PABPN1 ex- Total RNA from tissues, primary cell cultures and pression is likely due to distinct post-transcriptional fluorescence-activated cell sorting (FACS)-sorted cells mechanisms in different tissues. Taken together, our re- was isolated using TRIzol (Invitrogen, Carlsbad, CA) sults demonstrate that PABPN1 steady-state levels and according to the manufacturer’s protocol. Northern likely control of expression differ significantly in skeletal blotting was performed as described previously by muscle as compared to other tissues, which could have Ausubel et al. [19]. DNA probes were generated by important implications for understanding the muscle- polymerase chain reaction (PCR) using custom primers specific nature of OPMD. (PABPN1-F 5′-CCCAGGCAATGCTGGCCCAGTGAT CATGTCTC-3′ and PABPN1-R 5′-CTAGCCCGGCC Methods CCTGTAGATTCGACCCCGGGGC-3′,c-Myc-F 5′-GA Animals and primary muscle cell culture ACTTCACCAACAGGAACTATGACCTCG-3′ and c- Experiments involving animals were performed in ac- Myc-R 5′-GGTGTCTCCTCATGCAGCACTAGG-3′), cordance with approved guidelines and ethical approval primers from SA Biosciences, Valencia, CA (peroxisome from Emory University’s Institutional Animal Care and proliferator-activated receptor gamma coactivator 1α Apponi et al. Skeletal Muscle 2013, 3:23 Page 3 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 (PGC1α); PPM03360E, glyceraldehyde 3-phosphate de- Statistical analysis hydrogenase (GAPDH); PPM02946E) or by Ambion, Statistical analysis to determine significance between Austin, TX (QuantumRNA Classic II 18S). PCR products two groups was performed using a Student’st test. were labeled with [α- P]dCTP using a random primer One-way analysis of variance (ANOVA) was used for DNA labeling system (Invitrogen, Carlsbad, CA). comparisons between multiple groups as appropriate. All statistical analyses were performed using GraphPad Prism 5.0 for Macintosh (GraphPad Software). Differ- FACS ences were considered to be statistically significant at Mononucleated cells were enzymatically isolated from P <0.05. gastrocnemius muscles 3 days after BaCl injury and fluorescently labeled with antibodies to CD31 and CD45 Results (PE), Sca-1 (PE-Cy7), and alpha-7-integrin (AlexaFluor PABPN1 levels are lower in skeletal muscle compared to 649). Propidium iodide staining was used to gate out other tissues - - dead cells from the sort. Myoblasts (CD31 /CD45 /Sca- A better understanding of the mechanisms that underlie - + - + 1 /alpha-7-integrin /PI ) and non-myogenic cells (CD31 OPMD pathology can be obtained by analyzing the func- + + - - /CD45 /Sca-1 /alpha-7-integrin /PI ) were collected using tion of PABPN1 in skeletal muscle. To begin to identify a FACSAria II (Becton-Dickinson, Franklin Lakes, NJ). muscle-specific properties of PABPN1, we first examined Isolated cells were then processed for RNA extraction. the expression of PABPN1 across different tissues. Im- munoblot analysis revealed that PABPN1 steady-state levels vary significantly among mouse tissues, with skeletal Quantitative reverse transcription (RT)-PCR muscle displaying the lowest levels of PABPN1 (Figure 1A). cDNA synthesis from 100 ng RNA was performed using The low abundance of PABPN1 in skeletal muscle could M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). result from skewed misrepresentation of this protein mRNA levels were determined by real-time PCR using within the protein pool by the uniquely high levels of the iQ SYBR Green (Bio-Rad, Hercules, CA) and iCycler iQ Real-Time Detection System and software (Bio-Rad, Hercules, CA). The relative levels of PABPN1 were de- termined by the ΔΔCt method and normalized to the housekeeping gene HPRT1. Primers were from SA Bioscences, Valencia, CA (PABPN1: PPM25445A, HPRT1: PPM03559E). mRNA decay To analyze mRNA stability in vivo, mice were injected intraperitoneally with actinomycin D (Sigma-Aldrich, St. Louis, MO) at 2.5 μg/g and quadriceps muscles and kid- ney were collected 1, 2, 4 and 6 h later. To measure mRNA stability in primary myoblasts in vitro,5 μg/ml actinomycin D was added to the growth medium and cells were harvested 0.5, 1, 2 and 4 h later. Total RNA was extracted from tissues or cells and analyzed by north- ern blot and half-lives were determined by densitometry. 5′ and 3′ RACE In order to determine the 5′ and 3′UTRs of PABPN1 Figure 1 Nuclear poly(A) binding protein 1 (PABPN1) levels are transcripts, we used the 5′ and 3′ rapid amplification low in all skeletal muscles. Lysates prepared from different (A) of cDNA ends (RACE) system (Invitrogen, Carlsbad, mouse tissues (50 μg of total protein per lane), (B) mouse muscles CA), respectively. Total RNA from either muscle or (150 μg of total protein per lane) or (C) human tissues (20 μgof testis was used as a template according to the manu- total protein per lane) were immunoblotted with anti-PABPN1 antibody. Histone H3 and heat shock protein 90 (HSP90) were used facturer’s instructions. PCR products were cloned into as loading controls for mouse samples. Amido black staining was the pCR2.1 vector (TOPO TA cloning, Invitrogen, used as the loading control for human samples. Immunoblots are Carlsbad, CA) and sequenced by Beckman Coulter representative of at least three independent sets of tissues. Genomics, Danvers, MA. Apponi et al. Skeletal Muscle 2013, 3:23 Page 4 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 cytoplasmic proteins comprising the contractile machin- The 2.1 kb transcript, which was detected in all tissues ery in this tissue. However, relatively similar levels of both but was present at low levels in muscle (Figure 2B), uti- the nuclear protein histone H3 [20] and the cytoplasmic lizes a distal polyadenylation site 851 bp downstream of protein HSP90 [21] were observed between muscle and the stop codon (Figure 2A) [23]. The 1.4 kb represents other tissues, suggesting that the nuclear protein fraction the transcript that uses a proximal polyadenylation site is not under-represented in muscle. Furthermore, analysis 66 bp downstream of the stop codon (Figure 2A) [23]. of PABPN1 levels among different mouse muscles re- This 1.4 kb mRNA variant was the predominant tran- vealed even lower levels of this protein in the craniofacial script only in testis, but was also found in other tissues muscles (masseter, tongue and pharynx), some of which at much smaller amounts (Figure 2B). Interestingly, the are muscles primarily affected in OPMD patients [12], levels of the 1.4 kb PABPN1 transcript were very high in compared to other muscles of the body (Figure 1B). Sig- testis, which correlates with the very high levels of nificantly lower levels of PABPN1 in muscle as compared PABPN1 protein observed in this tissue (Additional file to other tissues were also observed in human samples 1: Figure S1). Furthermore, with the exception of testis, (Figure 1C), suggesting that the low levels of this pro- no significant variation in the ratio between the two tein in muscle are not species-specific findings, and mRNA isoforms was observed in the analyzed tissues. this may have physiologic implications for humans. Northern blotting also revealed a band of approximately To examine whether the expression of PABPN1 is reg- 3.6 kb that was observed in all tissues (data not shown). ulated at the protein or RNA level we performed north- This transcript was previously reported and suggested to ern blot analysis (Figure 2). This analysis revealed a be either a transcript of a related gene [22] or generated strong correlation between the low levels of PABPN1 by a distinct PABPN1 promoter [23]. We performed protein and the low abundance of PABPN1 transcript in both 5′RACE and 3′RACE from kidney, muscle and mouse skeletal muscle (Figure 2B), suggesting that con- testis but failed to identify any novel PABPN1 transcript trol of PABPN1 expression occurs at the RNA level, ei- other than the 1.4 kb and 2.1 kb mRNAs variants, ther by transcriptional or post-transcriptional means. As suggesting the 3.6 kb band might indeed represent a previously reported, PABPN1 has two major mRNA vari- transcript from a related gene. Together, our results ants, a 2.1 kb and a 1.4 kb transcript (Figure 2) [22,23]. from immunoblotting and northern blotting reveal low steady-state levels of PABPN1 mRNA and protein in skeletal muscle, which is indicative of either a decrease in PABPN1 transcription or altered mRNA stability in this tissue. PABPN1 levels are increased during muscle regeneration Adult skeletal muscle is comprised primarily of post- mitotic myofibers, however, it is a highly regenerative tis- sue that undergoes extensive repair after injury (Figure 3A) [24]. In the earliest phases of muscle regeneration, inflam- matory cells invade the tissue to remove dead tissue. Subsequently, large numbers of proliferative myoblasts derived from resident stem cells undergo differenti- ation and fusion to form new myofibers. Although PABPN1 levels are very low in adult muscle tissue, levels were significantly increased during the period of extensive cellular proliferation, differentiation and fusion that occurs 2 to 5 days after muscle injury (Figure 3B). However, 14 days after injury, when muscle architecture was restored (Figure 3A), PABPN1 levels were again low (Figure 3B). A similar pattern of upregulation was ob- Figure 2 Nuclear poly(A) binding protein 1 (PABPN1) mRNA served for the cytoplasmic poly(A) binding protein, levels are low in skeletal muscle. (A) Structure of the 2.1 kb and PABPC1 [2], during muscle regeneration, however the 1.4 kb PABPN1 transcripts (solid boxes represent coding regions and levels of the heat shock protein HSP90 remained constant open boxes non-coding regions). (B) Total RNA from different mouse tissues was analyzed by northern blot using a PABPN1 probe. over the time course. This result indicates that increased Two different exposures, short and long, are shown. 18S rRNA was levels of poly(A)-binding proteins during muscle regener- probed as a loading control. Figure is representative of at least three ation are not unique to PABPN1. To determine whether independent sets of tissues. the increased levels of PABPN1 observed at 2 to 5 days Apponi et al. Skeletal Muscle 2013, 3:23 Page 5 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 Figure 3 Nuclear poly(A) binding protein 1 (PABPN1) levels are increased during muscle regeneration in part due to increased levels in myoblasts. (A) Representative hematoxylin and eosin stained sections of gastrocnemius muscles at different times after BaCl injury are shown. (B) Lysates were prepared from gastrocnemius muscles at different times after injury and were immunoblotted with anti-PABPN1, PABPC1 or heat shock protein 90 (HSP90) antibodies (n = 3 per timepoint). (C) Total RNA was obtained from uninjured and injured muscle tissue (three independent samples) as well as fluorescence-activated cell sorting (FACS)-sorted myoblasts and non-myogenic cells obtained 3 days after muscle injury (pooled from five mice). PABPN1 mRNA levels were determined using real-time polymerase chain reaction (PCR) and hypoxanthine- guanine phosphoribosyltransferase (HPRT) mRNA was used as an internal control. Amount of PABPN1 mRNA relative to uninjured muscle is shown; n = 3. Data are mean ± SD; *P <0.05 vs uninjured muscle. after injury were due in part to myoblasts, we used flow all tissues but testis and contains a putative ARE, may be cytometry and specific antibodies to isolate myoblasts and subject to post-transcriptional regulation in different tis- non-myogenic cells (including inflammatory cells) from sues. To assess if this transcript is differently regulated in mouse muscles 3 days after injury. As we were unable to muscle, we analyzed PABPN1 mRNA stability in muscle perform immunoblots for PABPN1 on the small amount and kidney after blocking transcription in mice with acti- of cells isolated by flow cytometry, we used quantitative nomycin D. We observed that the half-life of the 2.1 kb RT-PCR to examine PABPN1 transcript levels in sorted PABPN1 transcript was significantly shorter in muscle cells compared to muscle tissue. Similar to what we ob- (2.3 h) compared to kidney (>6 h) (Figure 4A,E; served for PABPN1 protein, PABPN1 transcript levels Table 1). As a control to demonstrate similar transcrip- were increased approximately fivefold in injured com- tional inhibition between both tissues, we analyzed the pared to uninjured muscle (Figure 3C). We also found that stability of PGC1α and GAPDH mRNAs, known un- PABPN1 mRNA levels were exceptionally high in both stable and stable transcripts, respectively [25,26]. As - - - + sorted myoblasts (CD31 /CD45 /Sca-1 /alpha-7-integrin ) expected, PGC1α mRNA displayed a short half-life in + + + and non-myogenic cells (CD31 /CD45 /Sca-1 /alpha-7-in- both muscle and kidney (1.7 h and 2.6 h) compared to tegrin ) compared to uninjured muscle tissue (Figure 3C). GAPDH mRNA (>>6 h in both tissues) (Figure 4A,E; These data suggest that myoblasts significantly contribute Table 1). These results demonstrate a strong correl- to increased PABPN1 levels in regenerating muscle. We ation between the high steady-state levels of PABPN1 conclude that PABPN1 levels are not static in muscle but protein and the stable transcript in kidney, whereas in rather modulated by the physiologic state of the tissue, skeletal muscle, the low steady-state levels of PABPN1 suggesting a greater requirement for PABPN1 function dur- correlate with the unstable PABPN1 transcript. ing tissue repair. As shown earlier (Figure 3C), PABPN1 levels are mod- ulated during muscle regeneration and myoblasts con- PABPN1 mRNA is unstable in skeletal muscle tribute in part to the increased levels of PABPN1 during The two PABPN1 transcripts schematized in Figure 2A this process. We next investigated if the increase in arise from the usage of two different polyadenylation PABPN1 levels in myoblasts is accompanied by a corre- sites. The smaller 1.4 kb transcript contains virtually no sponding increase in PABPN1 mRNA stability. Similar 3′UTR, whereas the longer 2.1 kb transcript harbors a to myoblasts directly isolated from injured muscle, we 3′UTR of 851 bp containing a putative mRNA AU-rich found that cultured primary mouse myoblasts displayed destabilizing element (ARE) [23]. We hypothesized that the higher levels of PABPN1 mRNA compared to uninjured 2.1 kb transcript, which is the most abundant transcript in muscle tissue (Figure 4B). Consistent with this finding, Apponi et al. Skeletal Muscle 2013, 3:23 Page 6 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 Figure 4 Nuclear poly(A) binding protein 1 (PABPN1) mRNA is unstable in muscle tissue but stable in cultured myoblasts. (A) Total RNA was collected at different timepoints after injection of actinomycin D to inhibit transcription and PABPN1 mRNA decay was analyzed by northern blot. Time courses are shown for samples from muscle and kidney. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known unstable and stable transcript, respectively, were probed as controls (n = 3 per timepoint). To visualize PABPN1 signal in muscle samples, the blot was exposed significantly longer than for kidney samples. (B) Total RNA was obtained from skeletal muscle (SM) and cultured primary mouse myoblasts (Mb) and PABPN1 mRNA levels were determined using real-time polymerase chain reaction (PCR) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA was used as an internal control. The amount of PABPN1 mRNA relative to skeletal muscle (SM) is shown; n = 3 independent samples. Data are mean ± SD; *P <0.05 vs skeletal muscle. (C) Protein extracts were prepared from SM and Mb and immunoblotted with anti-PABPN1 antibody. HSP90 was used as a loading control. The immunoblot is representative of at least three independent samples. (D) Total RNA was collected at different timepoints after treatment of cultured primary mouse myoblasts with actinomycin D and PABPN1 mRNA decay was analyzed by northern blot; c-Myc and GAPDH, known unstable and stable transcripts in myoblasts, respectively, were probed as controls. Averages of densitometric measurements of northern blot bands were used to determine mRNA decay. The image is representative of at least three independent samples. (E) The decay profile of PABPN1 mRNA in muscle, kidney and cultured myoblasts plotted as mRNA amount relative to timepoint T = 0 h (n = 3 samples per timepoint). Data are mean ± SD. steady-state levels of PABPN1 protein were also higher in myoblasts present during muscle regeneration, is due at cultured myoblasts compared to muscle tissue (Figure 4C). least in part to the increase in PABPN1 mRNA stability in We analyzed the stability of PABPN1 transcripts in cul- those cells compared to muscle tissue. Taken together, our tured myoblasts. As observed in skeletal muscle tissue, the results suggest that PABPN1 expression in different 2.1 kb PABPN1 transcript was the predominant transcript tissues or during muscle regeneration is regulated by a in myoblasts (data not shown). However, in contrast to post-transcriptional mechanism that modulates tran- muscle tissue, the 2.1 kb PABPN1 transcript was ex- script stability. tremely stable in myoblasts (Figure 4D,E; Table 1). As expected, c-Myc mRNA, a known unstable transcript in Discussion myoblasts [27], had a short half-life compared to the Studying PABPN1 specifically in skeletal muscle is crit- much longer half-life for GAPDH mRNA, a known stable ical for defining the mechanisms which make this tissue transcript (Table 1). These results indicate that the high uniquely susceptible to the mutation causing OPMD. levels of PABPN1 in cultured myoblasts, and likely in Here, we report that steady-state levels of PABPN1 mRNA and protein are low in skeletal muscle and that Table 1 Nuclear poly(A) binding protein 1 (PABPN1) expression of PABPN1 in this tissue is controlled, at mRNA is unstable in muscle tissue but stable in cultured least in part, by post-transcriptional regulation of RNA myoblasts levels. We also demonstrate that PABPN1 levels are Muscle Kidney Myoblast modulated during muscle repair providing further sup- PABPN1 2.3 h >>6 h >>6 h port for regulation of PABPN1 expression in this tissue. PGC1α 1.7 h 2.6 h ND PABPN1 is not the only ubiquitous protein with a gen- GAPDH >>6 h >>6 h >>6 h eral function in basic cellular processes whose expression level is variable among tissues [28,29]. For example, the ex- c-Myc ND ND 0.3 h pression of histone H3A, transcription elongation factor GAPDH glyceraldehyde 3-phosphate dehydrogenase, ND not determined, PGC1α peroxisome proliferator-activated receptor gamma coactivator 1α. A1 (TCEA1) and heterogeneous nuclear ribonucleoprotein Apponi et al. Skeletal Muscle 2013, 3:23 Page 7 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 (hnRNP) C is relatively constant among different tissues, main 2.1 kb PABPN1 transcript expressed in skeletal whereas levels of GAPDH, β-actin and histone H2A are muscle harbors an ARE in the 3′UTR, we speculate this among the most variable within tissues [29]. These differ- pathway is a strong candidate for the control of PABPN1 ences in expression levels are most likely related to intrin- levels in skeletal muscle. sic properties of individual tissues and reflect differences in The specific PABPN1 expression pattern observed in metabolic activity and cellular structure. skeletal muscle may be an important feature that makes The extremely low levels of PABPN1 in skeletal this tissue more susceptible than others to the mutations muscle compared to other tissues may indicate a low re- in PABPN1 that cause the muscle-specific disease OPMD. quirement for this factor in basal muscle metabolism Whether the alanine expansion in PABPN1 leads to a and maintenance. Skeletal muscle is distinctly character- gain-of function or loss-of-function of this protein is un- ized by multinucleated, post-mitotic cells with a very known [12,15]. The nuclear aggregates observed in muscle specialized function and low complexity transcriptome of OPMD patients may exert toxic effects in the tissue as [30,31]. In skeletal muscle, a small number of genes con- hypothesized for other polyglutamine and polyalanine ex- tribute to a large fraction of the total mRNA pool, with pansion disorders [37-40]. However, as wild-type PABPN1 the ten most expressed genes in muscle accounting for can form reversible aggregates in neurons in response to 20% to 40% of the total mRNA [31]. The most abundant changes in cell physiology without overt pathology [41], transcripts in skeletal muscle encode proteins involved the toxicity of PABPN1 nuclear aggregates is unlikely to in contraction, glucose metabolism, ATP production and be the exclusive cause of OPMD etiology. In the loss-of ribosomal proteins [30,31], consistent with the role of -function model of OPMD etiology, one mechanism that this tissue in movement and metabolism. Such tran- could lead to a loss or decrease of PABPN1 function is an scripts encoding proteins involved in general cellular intrinsic reduction in PABPN1 activity caused by the ala- functions are usually stable with low turnover [27,32]. nine expansion. Although wild-type and mutant PABPN1 Therefore, the low complexity of the skeletal muscle appear to have similar polyadenylation activity in vitro [2], transcriptome associated with low turnover of a signifi- the effects of the alanine expansion on this or other cant fraction of its transcripts may explain why skeletal PABPN1 functions have not yet been addressed in the muscle has low requirements for a protein involved in context of skeletal muscle in vivo. Another mechanism mRNA metabolism such as PABPN1. that could explain a loss of PABPN1 function is the deple- Our data indicate the low levels of PABPN1 in skeletal tion of the soluble and functional fraction of PABPN1 by muscle are, at least in part, determined at the level of regulation of PABPN1 transcript stability. Regulation of mRNA decay rate is a key factor in determining the ex- pression pattern of many genes allowing rapid adapta- tion to changing cellular requirements [33,34]. PABPN1 levels increase significantly during skeletal muscle regen- eration suggesting a greater requirement for PABPN1 in myoblasts and non-myogenic cells such as inflammatory cells, which may be due to their highly proliferative sta- tus and to a more complex transcriptome compared to uninjured muscle tissue. As the increased levels of PABPN1 in regenerating muscle correlate with an increased tran- script stability in myoblasts and subsequent increase in the steady-state levels of PABPN1 transcript, we suggest that skeletal muscle employs a post-transcriptional mechanism to control PABPN1 levels according to the tissue requirements. Figure 5 Low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle may predispose this tissue to the mRNA decay rates are modulated by an interplay of deleterious effects of alanine-expanded PABPN1. We show specific stabilizing or destabilizing factors with the tran- muscle has lower levels of PABPN1 compared to other tissues in script, such as RNA-binding proteins and/or miRNAs normal individuals (N) but these levels are adequate for normal and their associated enzymes [35]. One of the most tissue function. In patients with oculopharyngeal muscular dystrophy studied post-transcriptional pathways is orchestrated by (OPMD), functional levels of PABPN1 could be decreased in all tissues due to expression of mutant PABPN1. However, muscle- a variety of RNA-binding proteins that interact with AU- specific pathology ensues in autosomal dominant OPMD because rich elements (ARE) within the 3′UTR of mRNAs the levels of PABPN1 fall below the threshold required to maintain [33,36] and many unstable mRNAs expressed in muscle proper tissue function. contain AU-rich elements in their 3′UTRs [27]. As the Apponi et al. Skeletal Muscle 2013, 3:23 Page 8 of 9 http://www.skeletalmusclejournal.com/content/3/1/23 sequestration in the nuclear aggregates present in muscle Authors' contributions LHA, AHC and GKP conceived and designed the study. LHA performed the of OPMD patients. In fact, a recent study that examined research. LHA, AHC and GKP analyzed the research and wrote the PABPN1 transcript levels in human muscle samples manuscript. All authors read and approved the final manuscript. reported a decrease in steady-state levels of PABPN1 mRNA after the fifth decade of life, the common age for Acknowledgements We thank Matthew Randolph for assistance with flow cytometry onset of OPMD symptoms [42]. This study also presented experiments. This work was supported by the Muscular Dystrophy evidence that this decrease in PABPN1 transcript levels is Association (MDA157523, MDA68022), and by the National Institutes of accelerated in OPMD patients [42]. These recent findings Health (NS059340, AR061987). support the idea that a loss of PABPN1 function could Received: 30 May 2013 Accepted: 28 August 2013 contribute to the muscle-specific pathology in OPMD. Published: 1 October 2013 Consistent with this idea, overexpression of wild-type PABPN1 reduces the pathology caused by the expression References 1. 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Skeletal MuscleSpringer Journals

Published: Oct 1, 2013

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