mt‐Ty 5'tiRNA regulates skeletal muscle cell proliferation and differentiation

Abstract In this study, we sought to determine the role of tRNA‐derived fragments in the regulation of gene expression during skeletal muscle cell proliferation and differentiation. We employed cell culture to examine the function of mt‐Ty 5’ tiRNAs. Northern blotting, RT‐PCR as well as RNA‐Seq, were performed to determine the effects of mt‐Ty 5’ tiRNA loss and gain on gene expression. Standard and transmission electron microscopy (TEM) were used to characterize cell and sub‐cellular structures. mt‐Ty 5’tiRNAs were found to be enriched in mouse skeletal muscle, showing increased levels in later developmental stages. Gapmer‐mediated inhibition of tiRNAs in skeletal muscle C2C12 myoblasts resulted in decreased cell proliferation and myogenic differentiation; consistent with this observation, RNA‐Seq, transcriptome analyses, and RT‐PCR revealed that skeletal muscle cell differentiation and cell proliferation pathways were also downregulated. Conversely, overexpression of mt‐Ty 5’tiRNAs in C2C12 cells led to a reversal of these transcriptional trends. These data reveal that mt‐Ty 5’tiRNAs are enriched in skeletal muscle and play an important role in myoblast proliferation and differentiation. Our study also highlights the potential for the development of tiRNAs as novel therapeutic targets for muscle‐related diseases.


| INTRODUCTION
Transfer RNAs (tRNAs) are essential components of the protein synthesis machinery. tRNA-derived small RNAs (tsRNAs) are a recently identified category of small non-coding RNAs (sncRNAs) that are generated by cleavage of pre-or mature tRNAs. 1 Although the nomenclature of tsRNAs has not yet been standardized, 2-4 they are mostly categorized into two groups: (1) tRNA halves (or tRNA-derived, stress-induced RNAs, referred to as tiRNAs) and (2) tRNA-derived fragments (tRFs).
tiRNAs are typically 30-50 nt long which is half the size of typical tRNAs. 1 As indicated by their name, tiRNAs have been found to be induced by stresses, such as arsenites, heat shock, and ultraviolet irradiation. 5 Angiogenin (ANG), a tRNA-specific ribonuclease, is required for stress-induced production of tiRNAs both in vitro and in vivo. 6 ANG cleaves tRNAs in the anticodon region, and leaves 2 0 -3 0 -cyclic phosphates at the 3 0 ends and hydroxyl groups at 5 0 ends of tRNAs 5 ; these are the unique characteristics of tiRNAs that differentiate them from tRFs. tiRNAs have been found to be involved in the regulation of protein translation 3,6 and mRNA stability 7 ; they also participate in multiple biological processes such as stem cell differentiation, 7 cell proliferation, 8 apoptosis 9 and immune response. 10 However, little is known about whether and how tiRNAs are involved in the regulation of skeletal muscle development.
Skeletal muscle development involves highly coordinated and complex molecular mechanisms and pathways, including myogenic regulator activation, 11 signalling transduction, 12 cell cycle arrest, 13 mitochondrial biogenesis, 14 and stress response. 15 Skeletal muscle differentiation is tightly accompanied by stress processes including reactive oxygen species production, DNA damage response, mitochondrial fission, and autophagic and mitophagic flux. 16 The continued identification and characterization of novel regulators of skeletal muscle development and function will be instrumental for the design of new therapeutic approaches for the treatment of muscle diseases.
In this study, our goal was to investigate the role of sncRNAs in skeletal muscle. We initially observed an enrichment of 5´tiRNAs in 2-month-old (2mo) mouse skeletal muscle, from which mt-Ty 5´tiR-NAs were identified to be the most abundant. Therefore, we inhibited or overexpressed mt-Ty 5´tiRNAs in the mouse skeletal muscle C2C12 myoblast cell line to assess their function. We determined that depletion of mt-Ty 5´tiRNAs led to reduced cell proliferation and differentiation, induced apoptosis and mitochondrial fragmentation. Correspondingly, the expression of genes involved in muscle cell growth and development was altered. These results demonstrated that precise regulation of the levels of mt-Ty 5´tiRNAs are required for muscle cell proliferation and differentiation.

| Cell culture
C2C12 myoblast cells (CRL-1772) were grown and maintained at 50% confluency in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and 100 units/ml penicillin and streptomycin. For transfection, cells were seeded at $60%-70% confluency; after 4 h, they were transfected with gapmers or tiRNA mimics using RNAiMAX (Thermofisher Scientific 13,778,150). The concentration for the treatment of gapmers and tiRNA mimics was 50 nM and 500 μM, respectively. Subsequent experiments including RNA-Seq and immunofluorescent staining were conducted after 24 h of transfection unless otherwise indicated. For experiments conducted using differentiating C2C12 cells, the cells were switched to a medium containing 2% horse serum to induce differentiation on the day that the cells reached 100% confluency; samples were collected on the indicated dates during differentiation. Myogenesis was monitored by staining cells with myogenic markers. Cells containing two or more nuclei were recorded as myotubes.

| RNA preparation and analysis
RNAs were extracted from tissue or cells using TRIzol (Thermofisher

| Plasmid DNA deep sequencing
Total RNAs from 2mo BL/6 mice were extracted as described above and analysed on a 15% denaturing polyacrylamide gel (PAGE) gel. RNA bands were stained by ethidium bromide (EtBr), and the area around 20-100 nt was cut and eluted with 0.3 M NaCl. Eluted RNA was ligated to a Linker with RNA ligase, then purified and linked to 3 0 adaptors. After another round of purification, reverse transcription was performed with the 3 0 antisense primer and PCR was performed with both the 5 0 and 3 0 primers. Finally, PCR products were used to quickly ligate using the TOPO system (Thermofisher Scientific). After transformation in ultracompetent cells, colonies were selected, and mini-preps were prepared for plasmid DNA deep sequencing.

| Near-infrared fluorescent northern blot and DIG northern blot
The mitochondrial tyrosine tRNA (mt-Ty) tRNAs and mt-Ty 5´tiRNAs in skeletal muscle of 2mo mice were determined by near-infrared fluorescent northern blot. 17 Briefly, 10-20 μg of total RNA from each sample was separated on 15% Urea-PAGE gel and transferred to Hybond N+ membrane (GE). The membrane was crosslinked twice using a 254 nm UV crosslinker at 120 mJ/cm 2 , which was followed by incubation with ExpressHyb hybridization solution (Takara) for 30 min at 30 C. The membrane was then hybridized overnight at 30 C with IR-dye conjugated probes (Table S1). To conjugate the probes with IR-dye, 2.5 nmol oligos were combined with 50 nmol IRDye 680RD (Li-Cor, catalogue number: 929-50005) or 800CW DBCO (Li-Cor, catalogue number: 929-55000) in PBS with a reaction volume of 50 μl, then incubated at room temperature in the dark for 6 h. After incubation, 2 volumes of AMPure XP beads (Beckman Coulter, catalogue number: A63881) and 5.4 volumes of isopropanol were mixed with the reaction to purify the IR-dye conjugated probes. The membrane was washed twice with 1X SSC-0.1% SDS buffer at room temperature on the following day and scanned on an Amershan Typhoon scanner (GE health) to detect emission at 600 nm and 800 nm.

| qPCR analysis
To determine changes in tiRNA levels, total RNAs were ran on a TBE-Urea gel and 10-50 nt bands were excised for RNA isolation. RNAs were reverse transcribed and amplified using the NCode miRNA amplification system (Thermofisher Scientific). qRT-PCR was performed to establish transcript levels of these and other downstream target genes discussed in the manuscript (primer source, IDT/Integrated DNA Technologies). Products were measured by absolute quantification and reported as a function of cycle threshold (Ct). mRNA expression was normalized to U6 expression, as reported in the text, thus obtaining relative expression (ΔCt) and mean fold change values (ΔΔCt). Following cycling, to ensure specificity, a melting curve analysis was carried out to verify the amplification of PCR products. The presence of one peak in the melting curve was employed as a requirement to ensure the absence of secondary products. Fold differences were calculated over control for each exposed group using normalized Ct values.
We trimmed adapter sequences and low-quality sequences from the RNA-Seq data using Trim Galore v0.6.6 (Martin 2011) with default parameters. The highquality reads were mapped to the mouse genome version mm10 using

| Mitochondrial morphology analysis
Analysis of mitochondrial network connectivity by immunofluorescent staining in individual cells was performed using the MiNA plugin in ImageJ. 18,19 2D microscopy images were pre-processed using the

| Quantification and statistical analysis
Quantification and statistical analysis for each experiment are detailed within each section of the figure legends. Graphpad Prism 6 Software was used to plot the graphs for all statistical analysis. Statistical significance was calculated using a t-test to compare two different groups.
Significance is defined as **p < 0.01 and ****p < 0.0001. For quantification of immunofluorescent experiments, n = 3 samples from three independent experiments. TEM images of mt were generated for n = 3 samples from three independent experiments. For quantification of surface area of mt and mitochondrial density, n = 128 mt for control gapmer treatment, n = 207 mt for mt-Ty gapmer treatment.

| Mt-Ty 5´tiRNAs are enriched in skeletal muscle
In the past, we have identified and investigated the expression and function of microRNAs (miRNAs) in skeletal muscle. 20,21 To identify additional sncRNAs in muscle, we harvested hind limb skeletal muscle tissue from 2mo BL/6 mice, extracted total RNA, and analysed the RNA using 15% denaturing PAGE. The area above the miRNAs ($ 21-23 nucleotide), and area corresponding to RNAs smaller than 45 nt, were excised from the gel and the RNA was eluted for cloning and sequencing, as described in the Materials and Methods.
DNA-sequencing of the cloned library and analysis revealed a list of sncRNAs, including mostly tiRNAs and ribosomal RNAs (rRNAs) which comprise 67% and 17%, respectively of the total sncRNA population, respectively ( Figure 1A). In eukaryotic cells, the nucleus and mt both encode tRNA genes that contribute to the production of cytoplasmic tRNAs and mitochondrial tRNAs (mt-tRNAs). 1 The detected tiRNAs are derived from both mitochondrial and cytoplasmic tRNAs, which occupy 24% and 43% of sncRNAs, respectively ( Figure 1A). The most highly enriched tiRNAs in skeletal muscle include Mitochondrial tyrosine (mt-Ty), mitochondrial cysteine (mt-Tc), mitochondrial valine (mt-Tv), and cytoplasmic Val/His/Asp/Gln/Glu/Gly/Lys 5´tiRNAs ( Figure 1B). The mt-Ty 5´tiRNA, mt-Tc 5´tiRNA, and Val 5´tiRNA are among the most abundant tiRNAs in skeletal muscle tissue ( Figure 1B). Notably, 5´tiRNAs with lengths of $26 and 31/32 nt are more abundant than tsRNAs of other lengths ( Figures 1B, C), suggesting that the predicted cleavage site targeted by ANG is located in the anticodon loop, as previously reported. 5 Moreover, 5´tiRNA and 3t iRNA expression levels are not equally distributed; 5´tiRNAs are more abundant than 3´tiRNAs ( Figure 1C), which is consistent with other published studies. 22,23 In the study by Dhahbi et al., 22 mt-Ty 5´tiRNA was demonstrated to be the most abundantly expressed tiRNA in skeletal muscle; therefore, we decided to make this the focus of our study. We examined the tissue distribution of mt-Ty 5´tiRNA expression in adult mice. We extracted RNAs from multiple tissues from 2mo mice, including the spleen, brain, lung, diaphragm, kidney, skeletal muscle, and heart.
TaqMan-based qPCR assays revealed that the mt-Ty 5´tiRNA was primarily expressed in skeletal muscle and heart but barely detectable in other tissues ( Figure 1D), suggesting a tissue-specific expression pattern.
Next, we performed near-infrared fluorescent northern blot 17 using probes targeting the mt-Ty 5´tiRNA to examine its expression from hind limb muscle tissues of mice at embryonic day 16 (E16), postnatal day 1 (P1), 1 mo, and 2 mo. mt-Ty 5´tiRNA is detectable only in the samples from 1mo and 2mo mouse skeletal muscle with much higher expression found in skeletal muscle of 2mo mice ( Figure 1E). (There is a slight shift of the bands from left to right as we allowed the gel bands to move further down to better resolve the different fragment sizes.) Notably, a comparable expression level of intact, mature mt-Ty tRNA was found in skeletal muscle during all the time points from E16 to 2mo ( Figure 1E).
These data suggest that the expression levels of mt-Ty 5´tiRNA and mt-Ty tRNA are independently regulated, consistent with that of prior reports. 5,7,8,24 Using C2C12 mouse skeletal muscle myoblasts we examined the expression of mt-Ty 5´tiRNA and other 5´tiRNAs during proliferation and differentiation. Cells were seeded at 50% confluency; the 5´tiRNA expression level was determined by qPCR using RNA isolated from a fraction of the cells collected during the proliferative state. The remainder of the cells (not used for the initial qPCR assay) were grown to confluency over the next 2-3 days; 5´tiRNA expression levels at 100% confluency were determined by qPCR as for the earlier samples. We found that the mt-Ty 5´tiRNA expression levels were substantially higher in C2C12 myoblasts at 100% confluency versus 50% confluency ( Figure 1F).
Subsequently, we differentiated C2C12 myoblasts into myotubes and compared 5´tiRNA expression levels in 100% confluent C2C12 myoblasts versus in myotubes. For this experiment, the C2C12 myoblasts were allowed to reach 100% confluency, then the growth media was changed to differentiation media (which contains 5% horse serum), and the C2C12 cells were allowed to differentiate into myotubes. We defined the day we changed media as differentiation day 0 (D0). Upon switching to differentiating medium, increased mt-Ty 5´tiRNA levels were detected in myotubes by differentiation day 5 (D5) ( Figure 1G). These data are consistent with the results demonstrating higher mt-Ty 5´tiRNA expression in skeletal muscle from 2mo mice.

| mt-Ty 5´tiRNAs regulate myoblast cell proliferation and differentiation
To investigate the function of mt-Ty 5´tiRNA in skeletal muscle, we designed an mt-Ty gapmer whose sequence is complementary to mt-Ty 5´tiRNA (Table S1) treated cells displayed a radial branching morphology and elongated appearance, whereas mt-Ty gapmer treated cells were irregular in shape, with some elongated cells and others with a more rounded appearance (Figure 2A, right panel). We calculated the cell number by staining the cells with Dapi and counting with ImageJ software. At this stage, the cells were at 70% confluency or slightly less, and the nuclei had not yet started to fuse; therefore, the cells were easily resolved using this method ( Figure 2C, D). We found that cell number was reduced by 34% upon mt-Ty gapmer treatment ( Figure 2B); To investigate the reason for the reduced cell numbers, we first examined the effect of mt-Ty 5´tiRNAs on cell proliferation. We stained Ctrl and mt-Ty 5 0 gapmer treated cells with the phospho-histone 3 (pH 3) and performed immunofluorescent imaging. PH3 is a proliferation marker which labels cells in mitosis and late G2 stages. 25 We treated C2C12   Figure 2D upper panel. We found that Ki67-positive cells dropped from 43% in Ctrl gapmer treated cells to 36% in mt-Ty 5 0 gapmer treated cells ( Figure 2D, lower panel). These findings indicate that gamermediated inhibition of mt-Ty 5´tiRNA reduces myoblast proliferation.  Figure 3A, B). Nuclear shrinkage is a typical feature of apoptosis 26 ; therefore, we measured nuclear size and found that nuclear size was significantly decreased in mt-Ty 5 0 gapmer treated cells ( Figure 3C). Therefore, our findings suggested that mt-Ty 5 0 gapmer treatment induced apoptosis.

| Depletion of mt-Ty 5´tiRNAs induced cell apoptosis and inhibited cell differentiation
Both apoptosis and proliferation are tightly associated with muscle differentiation and development 13,[27][28][29] ; therefore, we further examined the effects of mt-Ty gapmer treatment on C2C12 differentiation. We seeded C2C12 cells at 60%-70% confluency and transfected mt-Ty gapmers 6 h later. We allowed the cells to reach 90%-100% confluency (48 h after seeding). We changed the growth media to differentiation media when the cells were fully confluent, and we defined this time point as D0. We maintained C2C12 myoblasts in differentiation media for 3 days to allow C2C12 myoblasts to differentiate into myotubes. Then we examined the cells by immunofluorescent staining using the differentiation markers MYH and MF20 on differentiation day 3 (D3). During differentiation, the C2C12 cells remained as a monolayer, while the cells started to fuse together on the plate. A representative bright field image is shown in Figure 3D.
MYH and MF20 are antibodies that recognize different domains of myosin heavy chain; we used these antibodies to determine how mt-Ty 5´tiRNAs affect muscle development since myosin heavy chain is enriched in mature myotubes. 30 We found that myoblasts formed myotubes in Ctrl gapmer treated C2C12 cells where both MYH and MF20 were highly expressed ( Figure 3E, F, left panels). In contrast, myoblast differentiation and myotube formation were blocked in mt-Ty 5 0 gapmer treated cells, correlating with the low levels of MYH and MF20 staining ( Figure 3E, F, right panels). These results indicate that mt-Ty 5´tiRNA is required for C2C12 myoblast differentiation.

| mt-Ty 5´tiRNAs regulate skeletal muscle differentiation pathways
To gain a deeper understanding of the molecular function of mt-Ty 5t iRNA, we performed RNA-Seq analysis in C2C12 myoblasts that were treated with Ctrl gapmer or mt-Ty gapmer ( Figure S1A). We obtained high-quality data with a high mapping rate ($20 million reads per sample, >98% of high quality reads, and $ 85% total mapping rate) ( Figure S1B). Depletion of mt-Ty 5´tiRNA resulted in dysregulation of 1795 genes; among them, 494 genes were upregulated and 1301 genes were downregulated compared to Ctrl gapmer treated cells ( Figure 4A). Repressed genes were more abundant than activated genes when mt-Ty 5´tiRNA was depleted, suggesting that mt-Ty 5´tiRNA positively impacts gene expression. Among the most downregulated genes were Myog and Actc1, which encode the myogenic transcription factor myogenin and alpha cardiac muscle 1 actin, respectively ( Figure 4A); their downregulation is consistent with our data showing that inhibition of mt-Ty 5´tiRNA repressed myoblast differentiation. To validate our results, we performed qRT-PCR and found that several proliferation and differentiation genes including Myog, Pax7, Atoh8, Mod1, Actn3, Ttn, and Igfbp5 were significantly reduced, consistent with the results of the RNA-Seq ( Figure S2). GO-enriched terms for upregulated genes include cilium organization and assembly, axoneme assembly and microtubule-based transport, suggesting that loss of mt-Ty 5´tiRNA may enhance cell migration ( Figure 4B). In contrast, GO enriched terms for downregulated genes revealed that pathways related to 'muscle tissue development', 'striated muscle tissue development', and 'muscle cell differentiation' were among those with the most decreased levels ( Figure 4B

| Depletion of mt-Ty 5´tiRNAs induces mitochondrial fragmentation
Since mt-Ty was initially annotated as mitochondrial tRNA, we characterized the role of mt-Ty 5´tiRNA in this organelle. We stained proliferating C2C12 myoblasts with Tomm20, which stains the outer mitochondrial membrane, and imaged them using an Olympus  Figure S3B; p < 0.0001). There was no significant difference in mitochondrial mean branch length between the two groups ( Figure S3C). Mitochondrial volume, defined as the total signal from the mitochondrial footprint, was decreased significantly (p < 0.0001) from 16,273 counts/cell in Ctrl cells to 7284 counts/cell in mt-Ty gapmer treated cells ( Figure S3D).
Since quantification of mitochondrial parameters by MiNA suggested that 5 0 gapmer treatment induced obvious disruptions in mt ( Figure S3), we decided to further examine mitochondrial number and morphology by TEM. We found that mt in mt-Ty 5 0 gapmer treated cells were much smaller than that in Ctrl gapmer treated cells ( Figure 5C). Quantification of mitochondrial surface area showed that the average size of mt in mt-Ty 5 0 gapmer treated C2C12 myoblasts was 0.18 μm 2 , significantly lower than the 0.28 μm 2 value determined for control cells ( Figure 5D; p < 0.0001). We also determined the mt number in a specific area (per 1.1 μm 2 image field) to identify the mt density; we found that there were 3.2 and 6.4 mt per 1.1 μm 2 on average in Ctrl and mt-Ty 5 0 gapmer treated C2C12 myoblasts, respectively ( Figure 5E). To further examine whether the mitochondrial fragmentation observed was a result of mt-Ty 5´tiRNA inhibition, we performed GSEA analysis on genes related to mitochondrial organization. We found that genes associated with mitochondrial organization showed no significant change in gene expression in mt-Ty 5 0 gapmer treated cells ( Figure 5F); these data suggest that mitochondrial dysregulation is a secondary outcome from mt-Ty 5´tiRNA inhibition. Given that mt play an important role in the process of cell apoptosis, 31,32 evidence of mitochondrial fragmentation in mt-Ty gapmer treated cells further implicates mt-Ty 5´tiRNAs in the regulation of apoptosis in skeletal muscle.
3.6 | Ectopic expression of mt-Ty 5´tiRNAs affects expression of genes related to muscle development Next, we ectopically overexpressed mt-Ty 5´tiRNA in C2C12 myoblasts. Unexpectedly, we did not observe any obvious changes in cell morphology or cell number, and there was no significant change in cell proliferation by quantification of pH 3-positive cells (determined in mt-Ty 5´tiRNA transfected cells compared to control RNA, data not shown). Thus, we performed RNA-Seq analysis for RNAs extracted from C2C12 myoblasts transfected with Ctrl RNAs or synthetic mt-Ty 5´tiRNAs ($50 million reads per sample, >99% high quality reads, and $ 90% total mapping rate) ( Figure S1). Consistent with the lack of obvious changes in cell morphology and number, we did not observe a significant increase in differentially expressed genes and any gene expression changes were low (FC < =2). If we repeat our analyses with a slightly modified p-value (p < =0.05 and FC < =0.58), we detect 764 differentially expressed genes. In contrast to the results of our analyses of mt-Ty 5´tiRNA gapmer treated cells, overexpression of mt-Ty 5´tiRNA led to a greater number of upregulated genes than downregulated genes ( Figure 6A). Compared with controls, 536 genes were upregulated, while 228 genes were downregulated in samples where mt-Ty 5´tiRNA was overexpressed ( Figure 6A). Two of the genes that were enriched after overexpression of mt-Ty 5´tiRNA were Egr1 and Prl2c2. Egr1 (Early growth response 1) gene is a transcription factor which is required for differentiation of many cell types [33][34][35] ; Prl2c2 enhances cell growth and plays an important role in Yellow arrows mark the mitochondria. Scale bar =0.5 μm. (D) Surface area of mitochondria in Ctrl versus 5´gapmer treated C2C12 myoblasts was calculated using ImageJ (n > 100 mitochondria were randomly selected and quantified in each group). Data represent means ± SD. Statistical significance was calculated using a t-test to compare two different groups. ****p < 0.0001. (E) Mitochondria density was quantified as mitochondria number per 1.1 μm 2 area in TEM images of Ctrl or 5 0 gapmer treated C2C12 myoblasts (n = 36 square areas were randomly selected in each group for quantification). Data represent means ± SD. Statistical significance was calculated using a t-test to compare two different groups. ****p < 0.0001. (F) GSEA analysis of genes differentially expressed in mt-Ty 5 0 gapmer treated C2C12 myoblasts reveals no significant changes in mitochondrial organization-related genes. We also noticed that the 'skeletal muscle cell differentiation' category of genes was also upregulated, supporting a role for mt-Ty 5´tiRNAs in muscle differentiation ( Figure 6F). Collectively, overexpression of mt-Ty 5´tiRNA promoted the expression of a small set of genes which are critical for the regulation of cell growth and development. This corresponds well with our earlier observation that depletion of mt-Ty 5´tiRNAs led to inhibition of cell proliferation and differentiation.
Therefore, we propose that these data support a critical role for mt-Ty 5´tiRNAs in skeletal muscle function.

| DISCUSSION
Conditions, such as oxidative stress, nutritional deficiency, and hypoxia, have been shown to induce the expression of tiRNAs. 5,38,39 An increasing number of studies have also implicated specific tiRNAs in the promotion of cell proliferation 8 and cancer cell migration, 40 and the inhibition of stem cell pluripotency. 7 However, very few studies have investigated the role of tiRNAs in muscle development. We initially identified several tsRNAs including mt-Ty, mt-Tc, mt-Tv, and Val/His/Asp/Gln/Glu/Gly/Lys 5´tiRNAs that are highly enriched in F I G U R E 6 Ectopically expressed mt-Ty 5´tiRNAs in proliferating C2C12 cells promote expression of genes involved in muscle differentiation. (A) The volcano plot of gene expression changes in Ctrl RNA versus mt-Ty 5´tiRNA treated C2C12 myoblasts. Reduced genes (Fold Change <À1.5, pAdj <0.1) are coloured blue; activated genes (Fold Change >1.5, pAdj <0.1) are coloured red. (B) GO terms of activated (red) gene profile in C2C12 myoblasts with overexpression of mt-Ty 5´tiRNAs (pAdj). (C) GSEA analysis of genes differentially expressed in mt-Ty 5´tiRNA treated C2C12 myoblasts to compare their enrichment with repressed genes from 5 0 gapmer treated cells. (D) GSEA analysis of genes differentially expressed in mt-Ty 5´tiRNA treated C2C12 myoblasts to test their enrichment with activated genes from 5 0 gapmer treated cells. (E) GSEA analysis of genes differentially expressed in 5 0 gapmer treated C2C12 myoblasts to compare their enrichment with repressed genes from mt-Ty 5´tiRNA treated cells. (F) GSEA analysis of genes differentially expressed in 5 0 gapmer treated C2C12 myoblasts to test their enrichment with activated genes from mt-Ty 5´tiRNA treated cells.
2mo skeletal muscle; we decided to focus on mt-Ty 5´tiRNA since it is the most abundant of these molecules. We determined that mt-Ty 5t iRNAs were specifically expressed in skeletal muscle and heart; they also increased in abundance with age. This pattern of expression indicated that mt-Ty 5´tiRNAs could play important roles in muscle development. Therefore, we employed the muscle-derived C2C12 cell line to further investigate the functions of mt-Ty 5´tiRNA in muscle development.
To characterize the function of mt-Ty 5´tiRNA in muscle, we inhibited mt-Ty 5´tiRNAs in C2C12 myoblasts using gapmers.
Depletion of mt-Ty 5´tiRNAs led to a significant decrease in cell number, which was subsequently determined to be a result of decreased cell proliferation and induction of apoptosis. In addition, knockdown of mt-Ty 5´tiRNAs in C2C12 myoblasts prevented myotube formation in these cells even after 3 days of treatment to induce differentiation. Our RNA-Seq data also demonstrated that depletion of mt-Ty 5´tiRNAs altered expression of genes that are highly involved in proliferation, differentiation, and apoptosis.
Since these pathways are recruited during the induction of myoblasts into myotubes, these data provide insights into the molecular mechanisms by which mt-Ty 5´tiRNA knockdown is able to prevent this differentiation process.
Analysis of mitochondrial morphology revealed that their structure was disrupted in mt-Ty 5´tiRNA depleted cells; specifically, both mitochondrial size and number were decreased. Mitochondrial disruption can have severe adverse effects on cellular health since they are especially important for normal cardiac and skeletal myoblast function. [41][42][43][44][45] Not surprisingly, mitochondrial dysfunction often inhibits cell proliferation and causes cell death. 46,47 Therefore, we deemed it important to investigate whether the change in mitochondria is a direct result of mt-Ty 5t iRNA depletion. Our RNA-Seq analyses showed that mitochondrial organization and apoptosis gene pathways were not significantly altered in mt-Ty 5´tiRNA depleted cells; these data are consistent with a model in which the observed mitochondrial fragmentation resulted from apoptosis rather than causing it.
In this study, we also ectopically expressed mt-Ty 5´tiRNAs in C2C12 cells to determine if any changes would be induced. Although there was no obvious phenotype upon visual inspection, we determined that 536 genes were activated and 228 genes were repressed after mt-Ty 5´tiRNA overexpression; these data were in contrast to those determined following mt-Ty 5´tiRNA depletion, in which there were 1301 repressed genes, significantly more than the number of activated genes. To date, the primary molecular mechanism associated with mt-Ty 5´tiRNA function has been reported to be translational regulation. 3,6 It is intriguing that the activated genes we detected upon mt-Ty 5´tiRNA overexpression (Egr1, Snhg17, Prl2c2, and Errfi1) are involved in transcriptional regulation and are also required for cell proliferation and development. [33][34][35][36][37]48,49 The data we report here support a role for mt-Ty 5´tiRNAs as important regulators of gene expression that are enriched in muscle during development. We further propose that they function in muscle through the regulation of cell proliferation and differentiation pathways. possible, the data contained in these publications, as well as all other data generated by this project, will be deposited in other appropriate public repositories (e.g., Figshare).Data generated under this project will be administered in accordance with both the University of South Florida (USF) and NIH policies, including the NIH Data Sharing Policy and Implementation Guidance of March 5, 2003. We will adhere to the NIH Public Access Policy to ensure the timely release and sharing of final research data from NIH-supported studies for use by other researchers.
All published papers, abstracts, and proceedings will be available to the general public, including both the academic and industrial sectors.
Should any intellectual property arise which requires a patent, we will ensure the technology (i.e., materials and data) is available to the research community in accordance with the NIH Best Practices for the Licensing of Genomic Inventions and Section 8.

2.3, Sharing Research
Resources, of the NIH Grants Policy Statement. In other words, there will be no restrictions or limits placed on the sharing of data generated from this project. Material transfers will be made with no more restrictive terms than in the Simple Letter Agreement (SLA) or the Uniform Biological Materials Transfer Agreement (UBMTA) and without 'reachthrough' requirements.My laboratory has shown a commitment to sharing by providing cell lines, antibodies, plasmid constructs, and mice over the past 15 years. Any unique reagents that might be developed as a result of the research project will be made readily available to the scientific community. We will provide relevant protocols and published genetic and phenotypic data upon request. We will adhere to the NIH Grant Policy on sharing of unique research resources as outlined in the publication entitled "Sharing of Biomedical Research Resources Principles and Guidelines for Recipients of NIH Grants and Contracts".
"Other Research Resources" generated with funds from this grant may include DNA constructs and sequence data. These resources would be freely distributed upon request for non-commercial research. Any sequence data from bulk and single cell RNA-sequencing experiments will be deposited in a public database such as GEO upon publication.
We assume responsibility for distributing newly generated model organisms and we will fulfill these requests in a timely fashion. Following the characterization and peer-reviewed publication of mouse strains, they will be freely distributed to investigators at academic institutions wanting mice for non-commercial research. Recipient investigators will provide written assurance and evidence that the animals will be used solely in accord with appropriate IACAC review, that the recipient will not further distribute animals without our consent, and that animals will not be used for commercial purposes. To facilitate sharing and distribution of the transgenic mice and associated resources developed under this grant, mice will be maintained in a pathogen-free facility.