Long non‐coding RNAs are Transcriptional Regulators of Contractile Protein‐coding Genes in Skeletal Muscle

Abstract

Contractile properties of skeletal muscle determine strength, power and endurance. Muscle disuse and disease causes muscle atrophy and a shift in fiber‐type that diminish muscle strength and endurance. Better understanding of the transcriptional regulation of contractile protein genes by long non‐coding (lnc) RNAs may lead to development of promising new therapeutic treatment options. We previously identified and characterized four novel lncRNAs associated with key contractile protein‐coding genes by examining the transcriptome response to seven days of hindlimb unloading suspension (HS) in rat soleus (SOL) muscle using RNA‐Seq followed by validation with RT‐PCR. We showed that HS induced alterations in sense (S) and antisense (AS) lncRNAs associated with Myh7, Myl2, Tnni1, and Tnnc1. We hypothesized that these muscle disuse‐responsive lncRNAs regulate transcription of contractile genes by cis‐acting mechanisms. We sought to test this hypothesis using rat L6 myotubes to determine the cellular localization and function of the lncRNAs. We used strand‐specific Antisense Oligonucleotides (ASOs) in the form of Locked Nucleic Acid (LNA) GapmeRs to induce RNase‐H mediated degradation of lncRNAs. Real‐time PCR was used to assess RNA levels.Examination of nuclear and cytoplasmic RNA fractions revealed that each of the lncRNAs was <5‐fold higher in the nuclear fraction, indicating transcription‐mediated roles. We previously showed that an AS lncRNA at an intergenic location between Myh7 and Ngdn was upregulated during HS while Myh7 expression was decreased. Knockdown of this lncRNA resulted in a significant increase in Ngdn expression, suggesting that this lncRNA normally inhibits Ngdn. Thus, this lncRNA may serve to coordinate expression of Myh7 and Ngdn, which itself is implicated in translational regulation. Knockdown of a S lncRNA near TnnC1 resulted in elevated transcription of overlapping AS RNA and TnnC1 pre‐mRNA (NS). Further study is needed to determine how the lncRNA may regulate TnnC1. Gapmer‐mediated knockdown of AS lncRNA in the upstream‐promoter region of Tnni1 resulted in a 7‐fold increase in Tnni1 pre‐mRNA, suggesting that this AS lncRNA mediates transcriptional repression of Tnni1. Moreover, this AS lncRNA may hold potential as a potent and accessible target of therapeutic intervention in cases of disease or disuse where manipulation of slow‐myofiber specific Troponin I expression may be utilized to enhance contractile properties. In summary, we characterized novel lncRNAs thought to transcriptionally regulate expression of contractile genes in response to disuse. We conclude that transcriptional regulation of the examined contractile protein coding genes is influenced by associated lncRNAs.Support or Funding InformationMercer University School of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.