Characterization of Novel lncRNAs Transcribed Near Contractile Protein‐Coding Genes During Skeletal Muscle Unloading

Abstract

Unloading of skeletal muscle induces atrophy and a shift in slow fiber‐type muscles toward a fast‐fiber phenotype. This remodeling results in altered expression of contractile protein‐coding genes. It has become recognized that long noncoding RNAs (lncRNAs) are widely expressed throughout the genome and many have been shown to play a significant role in the transcriptional regulation of protein‐coding genes. We have identified four novel lncRNAs potentially involved in contractile phenotype shifts by examining the transcriptome response to seven days of hind‐limb unloading suspension (HS) in rat soleus (SOL) muscle using next generation sequencing data (RNA‐Seq) that was processed and analyzed with the Tuxedo Suite. A normal ambulatory group of rats served as controls (CON); (N=6). We hypothesized that these unannotated lncRNAs are differentially expressed during the phenotype shift and that they may regulate proximal protein‐coding genes. Transcription of the lncRNAs was examined with RT‐PCR and their abundance was assessed in both the sense (S) and antisense (AS) orientations, relative to the coding strand of the neighboring gene, by using gene‐specific primers during the RT and assessed at intervals of 3kb or less. The expression of these transcripts were characterized in CON and HS SOL through the use of both real‐time and endpoint PCR.We have identified lncRNAs near Myosin Heavy Chain 7/Slow Type 1 (Myh7), Myosin Regulatory Light Chain 2 (Myl2), Troponin I Type 1 (Tnni1), and Troponin C Type 1 (Tnnc1). There was a 38% decrease in the expression of Myh7 mRNA (p<0.05) with HS. We discovered an AS transcript overlapping Myh7 that extends to ~−19kb upstream of the Myh7 transcription start site (TSS) and was either increased (p<0.05) or unchanged in HS relative to CON SOL, depending on the target site. There is also an overlapping S lncRNA, but its expression averaged only 1/10th that of the AS lncRNA. In conjunction with an 84% HS‐induced decrease in Myl2 mRNA expression we discovered a S lncRNA that extends from −13kb relative to the Myl2 TSS and extends to the Myl2 coding region. It was decreased an average of 88% with HS at each interval measured. There was a single region 9kb upstream of Myl2 where transcription of AS RNA exceeded that of S RNA. At this site there was a 1.8‐fold increase in AS RNA in HS Sol (p<0.05). In regards to TnnC1, we report that there is a S lncRNA that extends ~5kb downstream from the protein‐coding region that is an average of 2.5 fold higher in HS (measured at ~1kb intervals). The expression of this lncRNA is coupled with a 4‐fold increase in Tnnc1 pre‐mRNA (p<0.05) and no change in mRNA levels. Upstream of Tnni1 we found varying levels of S and AS RNA. We found AS lncRNA that is transcribed from ~1.4 kb upstream of Tnni1 to ~8kb upstream. Its expression was 2.7‐fold higher in HS relative to CON. This was coupled with a 35% decrease in Tnni1 mRNA (not significant) and 52% decrease in pre‐mRNA (p=0.05) expression in HS Sol. In summary, we characterized four novel lncRNAs that are differentially expressed during muscle disuse. Further research is needed to determine if the reported increase in AS lncRNA regulates the observed decrease in protein‐coding mRNA expression.Support or Funding InformationMercer University School of Medicine and Navicent Health Foundation