And Post-Transcriptional Regulation of Rbm5 and Rbm10 in Mouse Cells As Evidenced by Tissue-Specific, Developmental and Dise

Gene 580 (2016) 26–36

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Research paper Co- and post-transcriptional regulation of Rbm5 and Rbm10 in mouse cells as evidenced by tissue-speciﬁc, developmental and disease-associated variation of splice variant and protein expression levels

Bartholomew Ozuemba a, Twinkle J. Masilamani b, Julie J. Loiselle b, Benjamin Koenderink c, Kaitlin A. Vanderbeck d, Jose Knee c, Céline Larivière b,d,LeslieC.Sutherlandb,c,e,f,g,⁎ a School of Life Science, University of Skovde, 541 28 Skovde, Sweden b Biomolecular Sciences Program, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada c AMRIC, Health Sciences North, 41 Ramsey Lake Road, Sudbury, ON, P3E 5J1, Canada d School of Human Kinetics, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada e Division of Medical Sciences, Northern Ontario School of Medicine, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada f Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada g Department of Medicine, Division of Medical Oncology, University of Ottawa, Ottawa, ON, Canada article info abstract

Article history: Background: Expression and function of the two RNA binding proteins and regulators of alternative splicing, Received 5 November 2015 RBM5 and RBM10, have largely been studied in human tissue and cell lines. The objective of the study described Accepted 31 December 2015 herein was to examine their expression in mouse tissue, in order to lay the framework for comprehensive func- Available online 16 January 2016 tional studies using mouse models. Methods: All RNA variants of Rbm5 and Rbm10 were examined in a range of normal primary mouse tissues. RNA Keywords: and protein were examined in differentiating C2C12 myoblasts and in denervated and dystonin-deficient mouse RBM5 skeletal muscle. RBM10 Gene expression Results: All Rbm5 and Rbm10 variants examined were expressed in all mouse tissues and cell lines. In general, Mouse Rbm5 and Rbm10 RNA expression was higher in brain than in skin. RNA expression levels were more varied be- Primary tissue tween cardiac and skeletal muscle, depending on the splice variant: for instance, Rbm10v1 RNA was higher in C2C12 skeletal than cardiac muscle, whereas Rbm10v3 RNA was higher in cardiac than skeletal muscle. In mouse Denervation brain, cardiac and skeletal muscle, RNA encoding an approximately 17 kDa potential paralogue of a small human RBM10 isoform was detected, and the protein observed in myoblasts and myotubes. Expression of Rbm5 and Rbm10 RNA remained constant during C2C12 myogenesis, but protein levels significantly decreased. In two muscle disease models, neither Rbm10 nor Rbm5 showed significant transcriptional changes, although significant specific alternative splicing changes of Rbm5 pre-mRNA were observed. Increased RBM10 protein levels were observed following denervation. Conclusions: The varied co-transcriptional and post-transcriptional regulation aspects of Rbm5 and Rbm10 expression associated with mouse tissues, myogenesis and muscle disease states suggest that a mouse model would be an interesting and useful model in which to study comprehensive functional aspects of RBM5 and RBM10. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Changes in RBP expression, with consequences for various RNA- related processes, are linked to many disease states including cancer, RNA Binding Proteins (RBPs) are key players in all aspects of RNA neurological disorders and muscular dystrophies (Lukong et al., 2008; metabolism including splicing, processing, stability and transport. Kim et al., 2009; Musunuru, 2003). RNA Binding Motif (RBM) proteins RBM5 and RBM10 are two structurally related proteins involved in the regulation of alternative splicing and, in the case of RBM10, mRNA sta- Abbreviations: D, day; Den, denervated; DT, dystonin-deﬁcient; kDa, kilodalton; PCR, bility (Sutherland et al., 2005; Bonnal et al., 2008; Fushimi et al., 2008; polymerase chain reaction; PND, post natal day; RBM, RNA Binding Motif; WT, wild-type. Jin et al., 2012; O'Leary et al., 2009; Sugliani et al., 2010; Wang et al., ⁎ Corresponding author at: AMRIC, Health Sciences North, 41 Ramsey Lake Road, Sudbury, ON P3E 5J1, Canada. 2013). Both RBM5 and RBM10 function to modulate apoptosis E-mail address: [email protected] (L.C. Sutherland). (Sutherland et al., 2000, 2005; Mourtada-Maarabouni et al., 2003;

Rintala-Maki and Sutherland, 2004; Wang et al., 2012; Oh et al., 2006). primary tissues, a myoblast differentiation model and hind limb skeletal Overexpression of exogenous RBM5 has also been shown to cause cell muscle from denervated and from dystonin-deficient mice. Our finding cycle arrest (Mourtada-Maarabouni et al., 2003; Oh et al., 2006). The of ubiquitous but tissue-, development- and disease-specific expression mRNA targets thus far identified for RBM5 encode the apoptosis modu- levels of Rbm5 and Rbm10 suggests that both genes play a necessary role lators CASP2 (Fushimi et al., 2008), FAS and FLICE (Bonnal et al., 2008), in all of the mouse cells examined, and that a mouse model would the deaminase AID (Jin et al., 2012), the cytoskeletal protein therefore be a useful tool for the study of functional consequences of DYSTROPHIN (O'Leary et al., 2009) and 11 spermatid developmental changes in their expression. As a consequence, our data provide a plat- regulators (O'Bryan et al., 2013). The mRNA targets thus far identified form for directed, Rbm5 and Rbm10 variant-specific functional studies for RBM10 encode the apoptosis modulator AT1 (Mueller et al., 2009) relating to myoblast differentiation, muscular atrophy and dystonin- and a number of developmental regulators associated with TARP syn- deficiency. drome (Wang et al., 2013). Thus far, RBM5 has been found to be expressed in a developmental 2. Materials and methods and tissue-specific manner. In general, higher levels are associated with reduced proliferation (consistent with a function that promotes cell 2.1. Animal work cycle arrest and apoptosis). For instance, in normal fibroblast cell lines and lymphocyte cultures, higher expression levels of RBM5 mRNA were All of the protocols pertaining to the procurement of mice tissue observed in samples collected from older versus younger people (Geigl samples were approved by the Laurentian University Animal Care et al., 2004) and in adult versus fetal thymus (Drabkin et al., 1999). Committee and followed stringent animal ethics guidelines (Canadian During seed maturation, expression of the RBM5 plant homolog Suppres- Council on Animal Care). All mice were strain CD1, purchased from sor of ABI3 (SUA) increased as dormancy was induced (Sugliani et al., the Charles River Laboratories (Saint-Constant, QC, Canada). For devel- 2010). RBM5 mRNA and variants encoding RBM5 + 5 + 6 and the anti- opmental and tissue-specific expression analyses in normal mouse tis- sense non-coding RBM5 AS1 (previously termed LUST) were higher in sues (brain, skin, cardiac and skeletal muscle), samples were excised muscle (heart and skeletal) and pancreas (Rintala-Maki and Sutherland, from Post Natal Day (PND) 7 and 14 mice. For expression analyses in 2009), demonstrating either tissue-specific or tissue-restricted (in the disease models, hind limb skeletal muscle was excised from two case of RBM5 AS1)expression.RBM5 was upregulated in drug resistant week-old (PND 14) dystonia musculorum (dtTg4) mice (Kothary et al., cancer cells that had undergone cell cycle arrest and experienced a 1988; Boyer et al., 2010) and wild type (wt) counterparts, and from 7- slow growth rate (Wang et al., 2004), and overexpressed RBM5 retarded day denervated (den) and sham-operated two month-old mice (Boyer tumor growth (Oh et al., 2006; Oh et al., 2002). On the other hand, re- et al., 2014). The denervation procedure was carried out by severing duced expression of RBM5 was observed in highly proliferating cells the hind limb sciatic nerve, and the sham-procedure was performed such as lung cancers, vestibular schwannomas and pancreatic ductal ad- by exposing the sciatic nerve without severing it, as previously de- enocarcinomas (Mourtada-Maarabouni et al., 2003; Oh et al., 2002; scribed (Boyer et al., 2014). Refer to Supplementary Table 1 for a break- Rintala-Maki et al., 2007; Welling et al., 2002; Peng et al., 2013), and down of experimental mouse tissue used. was one component of the 17-gene expression signature associated with solid tumor metastases in humans and mice (Ramaswamy et al., 2.2. Cell culture and differentiation 2003; Qiu et al., 2004). Far less is known about the expression of the RBM5 paralogue C2C12 and H9c2 cells were purchased from ATCC. Culture and differ- RBM10. Two predominant isoforms, RBM10v1 and RBM10v2, differing entiation were carried out as previously described (Masilamani et al., by a single 77 amino acid exon, are encoded by the human RBM10 2014; Menard et al., 1999). All cell culture medium, supplements and gene (Sutherland et al., 2005). A considerably smaller RBM10 isoform, reagents were purchased from Life Technologies (Burlington, ON, we refer to in humans as RBM10v3, has also been reported in GenBank Canada). (Accession Number AK000962.1). Often, data concerning RBM10 expression and function are restricted to RBM10v1. For instance, RBM10 2.3. RNA extraction was expressed in mid-gestation mouse embryos in the brachial arches and limbs (Johnston et al., 2010): nonsense or null mutations led to de- Total RNA extraction from brain, skin, cardiac and skeletal muscle velopmental anomalies, suggesting a key role during early embryogen- from CD1 mice and from wt, dt, sham and den hind limb muscles was esis (Johnston et al., 2010). Experimentally overexpressed RBM10 performed using the Qiagen RNeasy Kit (Qiagen, Mississauga, ON, increased apoptosis and reduced proliferation in rat vascular smooth Canada). Total RNA extraction from C2C12 cells was performed using muscle cells (Mueller et al., 2009). Functional analysis of both Tri-Reagent Solution (Molecular Research Centre Inc., Cincinnati, USA). RBM10v1 and RBM10v2 was carried out in cancer cell lines, where over- Total RNA was quantified using a NanoDrop 2000 UV–Vis spectropho- expression of each isoform induced apoptosis, and inhibition of either tometer (Thermo Scientific, Wilmington, USA) and the integrity was RBM10v1, specifically, or both RBM10v1 and RBM10v2, simultaneously, checked by visually assessing 28S, 18S and 5S band degradation follow- decreased sensitivity to the apoptogenic stimulus TNF-α (Wang et al., ing electrophoresis through a 2% agarose gel stained with SYBR® Safe 2012). In lung cancer tissue, nonsense, splicing and truncating muta- DNA Gel Stain (Life Technologies). tions throughout the RBM10 gene suggest that downregulation of this tumor suppressor gene-related protein is important to carcinogenesis 2.4. Reverse-transcription (RT) (Imielinski et al., 2012). Several lines of evidence suggest that both RMB5 and RBM10 play an All primers were purchased from Alpha DNA (Montreal, QC, Canada). important role in muscle cells since, as mentioned, the expression levels For tissues: 2 μg of total RNA from brain, skin, cardiac and skeletal muscle of RBM5 mRNA in human skeletal and cardiac muscle are elevated, and the hind limb muscles of wt, dt, sham and den mice, was reverse tran- RBM5 is involved in the regulation of dystrophin alternative splicing, scribed using oligo (dT) and Moloney Murine Leukemia Virus (MMLV) re- and upregulated RBM10 affects the proliferative and apoptotic potential verse transcriptase (Life Technologies). For the Rbm5 + 5 + 6 and Rbm5- of rat vascular smooth muscle cells. To better understand any potential AS1 strand-specific transcripts, DNase-treated total RNA was reverse muscle-related or developmental role of RBM5 and RBM10, an in vivo transcribed using the strand-specific primers mFactor XF: 5´ GTTCAAAT model would be optimal. To lay the groundwork for a mouse model ACTCTACTTGGTCC 3´ (for Rbm5 + 5 + 6)ormFactorXR:5´GATAGCCT for Rbm5 and Rbm10 function studies, we characterized the expression TTAAGATAAATGTA 3′ (for Rbm5-AS1) and Superscript II reverse tran- of both genes in a range of mouse tissues, including a selection of normal scriptase (Life Technologies). For C2C12 cells: total RNA was DNase 28 B. Ozuemba et al. / Gene 580 (2016) 26–36 treated before reverse transcription using oligo (dT) and MMLV. All In brain and skin, equal levels of Rbm5 and Rbm5 + 5 + 6 RNA were cDNAs were generated using standard procedures. noted, but significantly higher expression of Rbm5 + 6 (p = 0.01), Rbm5 AS1 (p = 0.0003) and both Rbm10v1 (p = 0.0001) and Rbm10v3 2.5. End-point polymerase chain reaction (PCR) (p = 0.0001) RNA was noted in brain compared to skin (Fig. 2B). When calculated as a fold-change, the differences in expression levels Quantification of mRNA expression levels of Rbm5 and Rbm10 vari- were greater than 10-fold for Rbm5 AS1 RNA, 60-fold for Rbm10v1 fi ants in mouse tissues and differentiating C2C12 cells was carried out RNA and 7-fold for Rbm10v3 RNA, but likely differing primer ef ciencies using end-point PCR. Primer sequences, annealing temperatures and preclude inter-variant comparisons. These data suggest that antisense amplicon sizes are listed in Supplementary Table 2. PCR conditions Rbm5 RNA and both isoforms of RBM10 may be of greater importance were 95 °C for 5 min, gene-specific cycle number at 95 °C for 30 s, inbrainthaninskintissue. primer-specific annealing temperature for 30 s and 72 °C for 60 s, In cardiac and skeletal muscle, differing expression levels of Rbm5, followed by a final extension at 72 °C for 10 min. As a negative control, Rbm5 + 5 + 6, Rbm5 AS1, Rbm10v1 and Rbm10v3 RNA were also ob- a no cDNA template was included. All the products were visualized in a served (Fig. 3B). Interestingly, Rbm10v1 RNA expression was higher in 2% agarose gel stained with SYBR® Safe DNA Gel Stain (Life Technolo- skeletal than cardiac muscle, whereas Rbm10v3 RNA expression was gies). All of the PCR reactions were performed in duplicate. Quantity higher in cardiac than skeletal muscle, suggesting that alternative splic- fi One ® software (Bio-Rad, Mississauga, ON, Canada) was used to quan- ing of Rbm10 pre-mRNA plays a role in tissue-speci c function and/or tify the band densities for the wt, dt, sham and den samples, whereas development and homeostasis. Once again, likely differing primer bind- AlphaEaseFC™ software (Alpha Innotech, Genetic Technologies Inc., ing kinetics precludes any inter-variant expression level comparisons. Miami, USA) was used to quantify band densities from the normal Since only one mouse had both cardiac and skeletal muscle extracted – fi mouse tissues and C2C12 cells. (animal F11 12: Table 1), statistical signi cance of the results could not be determined. 2.6. Real-time quantitative PCR (qPCR) 3.2. Expression of a 17 kDa RBM10 mouse isoform mRNA expression levels of Rbm5 variants in differentiating C2C12 End-point PCR directed at the detection of a mouse homolog of the cells was carried out, as previously described (Loiselle and Sutherland, small human RBM10 isoform that we refer to as RBM10v3 (Fig. 4A), 2014). Primer sequences, melting temperatures and amplicon sizes noted a consistently expressed amplicon of the anticipated size in cardi- are listed in Supplementary Table 2. The relative standard curve method ac muscle, skeletal muscle and brain (Fig. 4B). The human cDNA se- using the formula log10^((Cq (sample)–y intercept)/(−slope)) was quence (GenBank Accession Number AK000962.1) contains a 23 base- used for quantification (Larionov et al., 2005). The Hprt and Gapdh refer- pair exon 4 deletion, an exon 6 deletion and deletion of all sequence ence genes used for normalization were selected after validation using from the middle of exon 8 to the beginning of exon 22, compared to one-way ANOVA and NormFinder analyses (Masilamani et al., 2014). that of full-length RBM10. The potential protein encoded by this small transcript would have 100% homology to full-length RBM10 until the 2.7. Western blotting 23 bp exon 4 deletion, a 25 amino acid novel sequence following the deletion, a premature STOP codon within exon 4, and be approximately fi Quanti cation of RMB5 and RBM10 variant protein levels was car- 13 kDa in size. ried out using standard procedures, as previously described Using an anti-peptide RBM10 antibody raised against a 50 amino α (Sutherland et al., 2000). Primary antibodies used were mouse anti- - acid amino-terminal sequence (Fig. 4C), an ~17 kDa band was observed TUBULIN (1:10,000, sc-8035, Santa Cruz, U.S.A.), rabbit anti-RBM10 in the C2C12 cells following immunoblotting (Fig. 4D). This same band (HPA034972, Sigma Aldrich, U.S.A.), rabbit anti-RBM10 (A301-006 A, was also observed in the rat H9c2 myoblasts (Fig. 4D). Like the other “ ” Bethyl Laboratories Inc., U.S.A.), rabbit anti-RBM10 19 (18-003- two isoforms of RBM10 (RBM10v1 and RBM10v3), expression of this “ ” – 42019, Genway Biotech Inc., U.S.A.), rabbit anti-RBM10 20 (18 003- 17 kDa isoform gradually decreased between day 0 (D0) and day 7 42020, Genway Biotech Inc.) and LUCA-15-UK (Sutherland et al., (D7) of differentiation (Fig. 4D and E). Interestingly, expression was 2000). Secondary antibodies used were goat anti-mouse HRP- considerably higher in the H9c2 cells, which were established from em- conjugated (sc-2005, Santa Cruz, 1:20,000) and goat anti-rabbit HRP- bryonic BD1X rat heart tissue (Kimes and Brandt, 1976), compared to conjugated (sc-2004, Santa Cruz, 1:10,000). the C2C12 cells, which were established from mature C3H mouse skeletal muscle, following a crush injury (Yaffe and Saxel, 1977a). One 3. Results might be tempted to speculate that the 17 kDa RBM10 isoform is more highly expressed in cardiac than skeletal muscle; however, the 3.1. Expression of Rbm5 and Rbm10 mRNA in normal mouse tissue data presented in Fig. 4D and E, showing that expression in H9c2 myoblasts differentiating into either skeletal or cardiac myocytes is approx- We began our investigations by examining Rbm5 and Rbm10 expres- imately the same, suggests this is likely not the case. sion in normal mouse tissue. We had at our disposal various tissue sam- As shown in Fig. 4F, when expression was examined in the human ples from brain, skin, cardiac and skeletal muscle (Supplementary cervical carcinoma cell line, HeLa, the human lung adenocarcinoma Table 1). Expression of the murine equivalents of the known human cell line, A549, and the human lung transformed bronchial epithelial al- transcripts was examined semi-quantitatively using end-point PCR veolar cell line, BEAS-2B, no 17 kDa band was detected (even following with gene-specific primers, following strand-specific reverse transcrip- a much darker exposure, data not shown). This result suggests that the tion for variants Rbm5 + 5 + 6 and Rbm5 AS1. 17 kDa RBM10 isoform has a tissue-specific function that may be re- Transcripts encoding RBM5 and the four variants Rbm5Δ6, stricted to muscle and neurons (note that the human equivalent was Rbm5 + 5 + 6, Rbm5 + 6 and Rbm5 AS1,aswellasRBM10v1and isolated from “head” (neuronal tissue)). RBM10v3 (the murine equivalent of human RBM10v2) (Fig. 1), were Expression of the 17 kDa RBM10 isoform was also investigated detected in each of the tissue types (Fig. 2A, Fig. 3A). The fact that few using two additional, anti-peptide RBM10 antibodies raised against mice had more than one tissue type extracted limited tissue-specificex- internal sequences from exons 3 and 4 (“Genway 19”)orexon4 pression comparisons within animals, but we could perform analyses only (“Genway 20”). As shown in Fig. 4G, both antibodies were capable using five different mice that had both brain and skin samples extracted, of detecting the 17 kDa product in H9c2 cells. However, when an anti- and one mouse that had both cardiac and skeletal muscle extracted. peptide RBM10 antibody (from Bethyl), raised against a 50 amino acid B. Ozuemba et al. / Gene 580 (2016) 26–36 29

Fig. 1. RBM10 and RBM5 schematic detailing the various alternatively spliced transcripts found in humans. Forward and reverse primer-pairs used for the end-point PCR reactions (results depicted in Figs. 2 and 3), chosen based on human variants but designed using mouse-speciﬁc sequences, are represented by right and left pointing solid arrows for (A) RBM10 and (B) RBM5.

carboxy-terminal sequence, contained within only the two longer 48 h after induction of differentiation. As seen in Fig. 5A, using quantita- RBM10 isoforms (v1 and v3), was used, no 17 kDa product was ob- tive real-time PCR, with primers that would detect all sense Rbm5 tran- served (Fig. 4H). Peptide sequencing would be required to confirm scripts, the expression of Rbm5 RNA did not show any significant that the observed 17 kDa isoform, with homology to only the amino- increase in expression, and in particular, no spike between D1 and D2. terminal region of the full-length RBM10 protein, is indeed the homolog We therefore examined expression of TNF-α, and did not observe a of the small human RBM10 isoform. significant increase in expression following either 4 h or 10 h of induction of differentiation compared to time zero, as was previously reported (albeit using an RNase-protection assay) (Li and Schwartz, 2001). 3.3. Expression of Rbm5 and Rbm10 during C2C12 differentiation Protein expression was also monitored, at 24 h time intervals, over a differentiation period of seven days. Protein expression was shown to To provide the framework for potential future studies relating to significantly decrease by day 4 for RBM10v1 (p = 0.0038), day 6 for the function of RBM5 and RBM10 as developmental regulators in RBM5 (p = 0.001) and day 6 for RBM10v3 (p =0.0157),comparedto muscle, we examined expression during myogenesis using the day 0 (Fig. 5B). Taken together, these data suggest that neither RBM5 mouse C2C12 differentiation model. Background to this study in- nor RBM10 are key regulators of differentiation-associated cell cycle ar- cluded the following observations: RBM5 is highly expressed in rest, that in fact downregulation of RBM5 and RBM10 may be required human muscle (Sutherland et al., 2000; Drabkin et al., 1999), for myogenesis, and that this dowregulation regulation occurs post- overexpression of exogenous RBM5 in human cancer cell lines transcriptionally, since it is only apparent at the protein level. arrests cells in G0/G1, an event fundamental to differentiation (Mourtada-Maarabouni et al., 2003; Oh et al., 2006), expression of the cytokine TNF-α spikes just prior to the G0/G1 cell cycle 3.4. Expression of Rbm5 and Rbm10 RNA in disease states differentiation-associated arrest in C2C12 cells (Li and Schwartz, 2001), and there is a positive correlation between RBM5 and tumor Rbm5 and Rbm10 RNA and protein expression were examined in two necrosis factor alpha (TNF-α) expression in the human cancer cell muscle disease states. Firstly, we assessed the effect of hind limb muscle lines MCF-7 and Jurkat (Wang et al., 2012). Based on these observa- denervation, seven days after surgery, on the expression levels of Rbm5 tions, we anticipated that in the differentiating C2C12 cells there and Rbm10. Rbm5 RNA expression levels remained largely unaltered wouldbeanincreaseinRbm5,andperhapsRbm10, expression just (Fig. 6Ai) except for the upregulation of one transcript, Rbm5 + 5 + 6, prior to cell cycle arrest, between day 1 (D1) and day 2 (D2) in the muscle from denervated animals, compared to the sham controls (Dedieu et al., 2002), followed by a plateau. (p = 0.05). RBM5 protein data were not interpretable, since the anti- C2C12 cells were collected every hour for six hours following the in- body used for detection (Abcam ab85504) was subsequently found to duction of differentiation, then at 8, 10 and 12 h, and finally at 24 and be suboptimal (manuscript in preparation). Rbm10 expression at the 30 B. Ozuemba et al. / Gene 580 (2016) 26–36

Fig. 2. Expression of Rbm5 and Rbm10 RNA in mouse brain and skin tissue. (A) In brain and skin tissue from wild-type CD1 mice (littermates and non-littermates from various breeding pairs), RNA expression levels were examined following end-point RT-PCR. Note, a brain and a skin sample were examined from each mouse. (A) Agarose gels, stained with SYBR® Safe, showing PCR amplicons representing the indicated transcripts. (B) Graphs capturing the standardized and β-actin-normalized gel densitometric data. Error bars represent ± SEM, analyzed in technical duplicate. Statistical signiﬁcance was determined using an unpaired Student's t-test.

RNA level remained unchanged (Fig. 6Ai); however, at the protein level, Rbm5 + 6 (p = 0.02), was noted. No overall change in Rbm5 total RNA both RBM10 isoforms were significantly upregulated in the denervated expression was observed. These data suggest that, in muscle, dystonin tissue compared to the sham control tissue (p =0.03forRBM10v1and expression is unrelated to that of RBM10, but may be related to the reg- p = 0.02 for RBM10v3) (Fig. 6Aii), although it should be noted that RNA ulation of Rbm5 pre-mRNA alternative splicing. and protein were not extracted from the same mouse cohort. Taken together, these data suggest that alternative splicing of Rbm5 pre-mRNA 4. Discussion remains largely unchanged in skeletal muscle as a result of interruptions in neuromuscular activity, whereas RBM10 protein stabilization can be 4.1. Tissue-specificregulation significantly influenced by neuromuscular inactivation. Secondly, we examined expression of Rbm5 and Rbm10 RNA in hind This body of work constitutes the first comprehensive characteriza- limb skeletal muscle of dystonin-deficient (dtTg4) mice, which exhibit tion of Rbm5 and Rbm10 expression in mice. What was revealed was an various muscle abnormalities (Dalpe et al., 1999). Whereas Rbm10 RNA animal model in which RNA expression - of the myriad of Rbm5 and expression was not significantly changed in the dystonin-deficient com- Rbm10 variants - appears to be ubiquitous. Though ubiquitous, we did paredtothewild-type(wt)muscletissue(Fig. 6B), a significant down- observe differences in the level of expression, suggesting tissue- regulation of the two Rbm5 RNA variants, Rbm5Δ6 (p = 0.01) and specific regulation. Most notably, overall transcription levels of both B. Ozuemba et al. / Gene 580 (2016) 26–36 31

Fig. 3. Expression of Rbm5 and Rbm10 RNA in mouse cardiac and skeletal muscle tissue. (A) In cardiac and skeletal muscle tissue from wild-type CD1 mice (littermates and non-littermates from various breeding pairs), RNA expression levels were examined following end-point RT-PCR. (A) Agarose gels, stained with SYBR® Safe, showing amplicons representing the indicated transcripts, following RT-PCR. (B) Comparison of transcript expression levels between cardiac and skeletal muscle tissue, from one mouse (F11–12). Graphs capture the standardized and β-actin-normalized gel densitometric data.

Rbm5 and Rbm10 were higher in brain compared to skin (Rbm5 =3- 4.2. Developmental regulation fold, Rbm10 = 11-fold) (Fig. 2B), and higher in cardiac compared to skeletal muscle (Rbm5 =4-fold,Rbm10 = 2-fold) (Fig. 3B). With The C2C12 cell line was established from skeletal muscle satellite regards to the expression of variants of one gene, we also noted that cells from a C3H mouse, following a thigh muscle crush injury (Yaffe the ratio of Rbm10v1:Rbm10v3, at the transcript level, was lower in car- and Saxel, 1977b). Differentiation of C2C12 cells into myotubes, follow- diac than in skeletal muscle (an observation consistent with a previous ing partial serum withdrawal, is a well characterized model of report relating to lineage-specific expression patterns in differentiating myogenesis that mimics the in vivo process, involving a series of chang- rat H9c2 cells) (Fig. 3B), suggesting that alternative splicing of Rbm10 es that includes cell cycle arrest, cell elongation and cell fusion, accom- pre-mRNA is regulated in a tissue specific manner. Likewise, expression panied by apoptosis in specific subsets of cells (Burattini et al., 2004; levels of the two transcripts, Rbm5 + 6 and Rbm5 AS1,weresignificantly Schoneich et al., 2014). higher in brain compared to skin (Fig. 2B), suggesting that truncation of Cell cycle arrest occurs in C2C12 cells within 48 h of serum with- RBM5 (from Rbm5 + 6), and expression of a long non-coding antisense drawal (Kaspar and Dvorak, 2008), while apoptosis, though necessary Rbm5 RNA (from Rbm5 AS1), are likely more functionally active in neu- in a subset of cells during the induction stage, is predominant in differ- ronal than epithelial cells. entiated myotubes (Schoneich et al., 2014). The fact that no significant 32 B. Ozuemba et al. / Gene 580 (2016) 26–36

Fig. 4. Expression of a 17 kDa RBM10 isoform. (A) Diagram of human RBM10v3, and the anticipated mouse orthologue following end-point PCR with the primers indicated by a forward and reverse arrow. (B) RNA expression of the expected 127 base-pair amplicon, in mouse cardiac, skeletal and brain tissue from various breeding-pairs and litter-mates. (C) Diagram of RBM10, with 24 exons, showing the regions used for anti-peptide antibody generation. Each peptide sequence was 50 amino acids in length (diagram not drawn to scale). (D) Protein expression, using an RBM10 antibody from Sigma, at a 1:1000 dilution, in differentiating C2C12 and H9c2 cells. (E) Protein expression in C2C12 cells, using the RBM10 Sigma antibody at a 1:500 dilution, and in H9c2 cells, using the RBM10 Sigma antibody at a 1:10,000 dilution. (F) Protein expression in the cell lines indicated, using the RBM10 Sigma antibody at a 1:500 dilution. (G) Protein expression in H9c2 cells using two different RBM10 antibodies from Genway, “19” which is able to detect any isoform with either exon 3 or exon 3 and exon 4 sequence, and “20” which is able to detect any isoform with exon 4 sequence. (H) Protein expression in C2C12 cells using two different RBM10 antibodies, the Genway 20 and a Bethyl.

increase in Rbm5 RNA expression levels was observed within either the may not involve regulation of either the cell cycle or apoptosis. The ﬁrst 48 h of differentiation or during the ﬁnal stage of differentiation gradual decrease in expression of both RBM5 and RBM10 protein levels, (Fig. 5) suggests that, contrary to what has previously been reported between D0 and D7 of differentiation, which is also observed during dif- in cancer cell lines, the functional role of RBM5 during myogenesis ferentiation of rat H9c2 cells (Loiselle and Sutherland, 2014), does B. Ozuemba et al. / Gene 580 (2016) 26–36 33

Fig. 5. Expression of Rbm5 and Rbm10 RNA and protein during C2C12 myoblast differentiation. (A) C2C12 cells were collected 24 h pre-induction of differentiation, immediately following induction of differentiation (D0), every hour for six hours following the induction of differentiation, then at 8, 10 and 12 h, and finally at 24 and 48 h after induction of differentiation. Rbm5 RNA expression was monitored using real-time PCR, and presented as a fold-change relative to D0. Expression was normalized to the geometric mean of Gapdh and Hprt RNA expression. Error bars represent ± SEM from three biological replicates, performed in technical duplicate. (B) RBM5 and RBM10 protein expression, monitored by Western blotting, from 24 h time points ranging between D0 and D7, using the LUCA-15-UK (RBM5) and Bethyl (RBM10) antibodies. One representative Western blot, from five biological replicates, is shown for each full- length protein. Graphs capture the densitometric data from at least technical duplicates of each of the five biological replicates, standardized to a ladder/protein marker band and normalized to α-tubulin, as a fold-change compared to D0. Error bars represent ± SEM. Statistical significance, relating to changes in expression levels relative to D0, was calculated using an unpaired Student's t-test, with *p b 0.05, **p b 0.01 and ***p b 0.0001. suggest that the two genes encode products that are required for the ini- 4.3. Novel small Rbm10 isoform that is highly expressed in rodent tial stages of differentiation only, but what these functions are remains myoblasts to be determined. The lack of correlation between Rbm5 and Rbm10 RNA and protein Our identification of a 17 kDa isoform of RBM10 is the first report of levels during differentiation suggests that protein expression is regulat- the expression of a potential human small RBM10 homolog in a rodent. ed post-transcriptionally. It is well documented that microRNAs Our observation that expression, of not only RBM10v1 and RBM10v3 (miRNA) are involved in myogenesis and can either repress mRNA but also the 17 kDa isoform, decreases during myogenesis (Figs. 4Eand translation or lead to mRNA degradation (Ge and Chen, 2011; 5B), suggests that irrespective of the underlying post-transcriptional in- Dmitriev et al., 2013). For instance, expression of the neuronal hibitory mechanism, it is not variant/isoform specific. polypyrimidine tract-binding protein, nPTB, is reduced during C2C12 differentiation following translational repression by miR-133, with no accompanying reduction in mRNA expression levels (Boutz et al., 4.4. A neuronal connection? 2007). While no miRNA inhibitor of RBM10 has been identified, it is possible that miR-660 is responsible for the downregulation of RBM5 in dif- What we learned from the expression data was, firstly, that tran- ferentiating C2C12 cells: previous work using RNA from human affinity- scription of both Rbm5 and Rbm10 was significantly higher in PND 14 purified CD56+ myoblasts demonstrated a reduction in RBM5 RNA ex- mouse brain compared to skin. This observation was not entirely unex- pression by miR-660 (Dmitriev et al., 2013). In our C2C12 experiments, pected, since brain cells have a substantially slower rate of proliferation only RBM5 protein was shown to be downregulated during differentia- than skin cells, and expression of RBM5 is usually associated with de- tion, not Rbm5 RNA. creased proliferation or senescence (Sugliani et al., 2010; Oh et al., 34 B. Ozuemba et al. / Gene 580 (2016) 26–36

Fig. 6. Expression of Rbm5 and Rbm10 RNA and protein in denervated muscle and dystonin-deficient muscle. (A) Sham versus denervated (Den) muscle, seven days following denervation. (i) The RNA expression levels, normalized to β-actin,ofRbm5 + 5 + 6 in denervated mice. Error bars represent ± SEM from N = 7 (Sham) and N = 4 (Den). Significance was calculated using an unpaired Student's t-test, where *p = 0.05. (ii) The protein expression levels of RBM10v1 and RBM10v3, from Western blotting, normalized to a band on the Ponceau-stained membrane. Data plotted from densitometric analysis of bands from N = 8 different mice. Significances calculated using an unpaired, two-tailed t-test, and reported in the text. (B) Wild-type versus dystonin-deficient (dt) muscle. The RNA expression levels of Rbm5Δ6 and Rbm5 + 6, normalized to β-Actin. Error bars represent ± SEM from N = 4 (wt) and N=6(dt). Significance was calculated using an unpaired Student's t-test, where *p b 0.05, **p =0.01.

2006; Geigl et al., 2004; Wang et al., 2004). Secondly, expression of the suggests that Rbm5 AS1 expression does not inhibit full-length Rbm5 long non-coding (lnc) antisense Rbm5 transcript, Rbm5 AS1,wasupreg- RNA expression in the mouse brain. Thirdly, consistent expression of ulated in PND 14 mouse brain compared to skin, a finding consistent the mRNA encoding a 17 kDa RBM10 isoform was observed in brain with previous evidence that many lnc antisense transcripts are and muscle tissue. The fact that the potential human paralogue of this expressed in neurons, where they regulate sense transcript expression gene was isolated from “embryo, mainly head” (GenBank Accession (Szafranski et al., 2015). The observation that both Rbm5 and Rbm5 Number AK000962.1), with expression being totally absent from two AS1 RNA are higher in PND 14 mouse brain compared to skin, however, human cancer cell lines (cervical and lung) and a non-cancerous but B. Ozuemba et al. / Gene 580 (2016) 26–36 35 transformed human cell line (lung) (Fig. 4F), suggests that this RBM10 observations in the rat H9c2 in vitro model. This finding reinforces the isoform has restricted expression and function, which may be usefulness of an in vivo mouse model for further physiologically relevant neuromuscular. functional studies. Finally, the upregulation of RBM10 protein expression in denervated Supplementary data to this article can be found online at http://dx. tissue, compared to sham (Fig. 6Aii), reinforces a possible functional doi.org/10.1016/j.gene.2015.12.070. role related to the neuromuscular activity associated with muscle atrophy or atrophy-related regeneration. While muscle atrophy, a state as- Acknowledgments sociated with decreased cell size and loss of sarcomeric proteins (Midrio, 2006), can occur as a result of muscle injury, it is also a serious This work was funded by NSERC Grant #9043429 to L.C.S., an Alex- component of many disease states (including cancer, AIDS, sepsis and ander Graham Bell Canada Graduate Scholarship-Masters to J.J.L., an diabetes), inactivity and aging. Since RBM5 and RBM10 are known reg- NSERC Vanier Award to T.J.M. and a Laurentian University Research ulators of apoptosis, and apoptosis occurs in atrophying denervated Fund grant to C.L. muscle (Allen et al., 1997; Dupont-Versteegden et al., 1999), we anticipated increased levels of RBM5 and RBM10 in the denervated muscle samples (Sandri, 2008; Boudriau et al., 1996). The fact that only References RBM10 protein (Fig. 6Aii) but not Rbm10 RNA (Fig. 6Ai) was affected Allen, D.L., Linderman, J.K., Roy, R.R., et al., 1997. Apoptosis: a mechanism contributing to in the denervated tissue suggests that the regulation of Rbm10 in remodeling of skeletal muscle in response to hindlimb unweighting. Am. J. Physiol. atrophied or regenerating muscle occurs post-transcriptionally, and in- 273, C579–C587. volves an increased rate of translation and/or protein stabilization. With Bonnal, S., Martinez, C., Forch, P., Bachi, A., Wilm, M., Valcarcel, J., 2008. RBM5/Luca-15/ H37 regulates Fas alternative splice site pairing after exon definition. Mol. Cell 32, regards to Rbm5, one alternative splicing difference in Rbm5 pre-mRNA 81–95. was noted in the denervated tissue compared to the sham control Boudriau, S., Cote, C.H., Vincent, M., Houle, P., Tremblay, R.R., Rogers, P.A., 1996. Remodel- (Fig. 6 Ai), but the overall expression level of the affected variant, ing of the cytoskeletal lattice in denervated skeletal muscle. Muscle Nerve 19, fi 1383–1390. 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