Muscle aging-related IncRNA LANCLI-AS1in myogenesis and muscle regeneration

National Institute On Aging

Investigators

Abstract

LANCL1-AS1 levels decline with muscle aging, increase with human myogenesis By combining the analysis of RNAs expressed in a human cultured model of myogenesis, as well as sequencing and muscle aging biopsies (GESTALT), we identified 48 differentially expressed RNAs shared with these analyses; 5 of these are lncRNAs. Many lncRNAs were shown to correlate with myogenesis and muscle regeneration, but few have known function. We found that LANCL1-AS1 was the most differentially expressed lncRNAs in this combined analysis. Moreover, LANCL1-AS1 is the second most significantly downregulated RNA by negative binomial models analysis during muscle aging. In addition, the linear regression plots showed that LANCL1-AS1 levels were significantly reduced with advancing age. RT-qPCR analysis confirmed a rise in the levels of LANCL1-AS1 during the early stages in human myogenesis, and continuous upregulation of OIP5-AS1 with advancing myogenic differentiation. Those results suggest that LANCL1-AS1 may play an important role for myogenesis, muscle regeneration and muscle aging. LANCL1-AS1 may promote myogenesis, translocates to cytoplasm during myogenesis To begin to study the role of LANCL1-AS1 in myogenesis, we silenced LANCL1-AS1 in AB678 human myoblasts. interestingly, this intervention reduced myogenesis progression in human myoblasts, as determined by measuring myotube formation. To further understand the potential mechanism by which LANCL1-AS1 affected myogenesis, we fractionated RNA through sucrose gradients. LANCL1-AS1 translocated from the nucleus to the cytoplasm during myogenic differentiation, suggesting that LANCL1-AS1 may play the different roles during myogenesis by targeting with different molecular partners on different subcellular spaces. Since the binding partner is critical for lncRNA functions, we used biotinylated antisense oligonucleotides (ASO) RNAs to pull down interacting RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP and RT-qPCR analysis revealed that LANCL1-AS1 was very efficiently pulled down by specific LANCL1-AS1 ASOs. Recently, a few examples have been reported of lncRNAs that can be translated partially into small protein products (micropeptides). Interestingly, we also found LANCL1-AS1 is present in small polysome fractions, suggesting that LANCL1-AS1 has protein-coding potential. Notably, differentiated myoblasts have more LANCL1-AS1 is in the heavy polysome fraction, suggesting that LANCL1-AS1 may have functional mcicropeptide during myogenic differentiation. Ongoing Efforts AIM 1: to determine if muscle aging related-lncRNA LANCL1-AS1 promotes myogenesis. By combining the analysis of RNAs expressed in a human cell cultured model of myogenesis, and in human muscle aging biopsies (GESTALT), LANCL1-AS1 was found as the most differentially expressed lncRNA. Moreover, LANCL1-AS1 was the second most significantly downregulated RNA by negative binomial model analysis in muscle aging. RT-qPCR analysis of cultured myoblasts revealed a rise in LANCL1-AS1 levels during early stages in human myogenesis, and continuous upregulation of LANCL1-AS1 with advancing myogenic differentiation. In addition, linear regression plots showed that LANCL1-AS1 levels declined significantly with advancing age. These results were validated in human muscle biopsies by RT-qPCR analysis. Although LANCL1-AS1 was expressed in nerve cells, its levels did not increase with neural differentiation, suggesting that LANCL1-AS1 increases specifically in myogenesis. Our preliminary results also showed that silencing LANCL1-AS1 attenuated myogenesis, as determined by monitoring myotube formation and by measuring creatine kinase activity. AIM 2: LANCL1-AS1 interacts with mitochondria RNA-binding protein LRPPRC and regulates muscle contraction related genes as well as mitochondria encoding genes. Given that the function of lncRNAs is closely linked to their interacting partners, we identified LANCL1-AS1-interacting protein by ChIRP-Mass Spectrometry analysis. Among them, the top candidate was LRPPRC, a nuclear and mitochondrial RNA-binding protein (RBP), which regulates metabolic pathways and mitochondria homeostasis by driving transcriptional and post-transcriptional programs. We thus validated the interaction between LANCL1-AS1 and LRPPRC by ChIRP-Western and CLIP assays. Silencing LRPPRC attenuated myogenesis, and RNA-sequencing analysis revealed that loss of LANCL1-AS1 reduced muscle contraction, ion exchange activity, and the expression of many mitochondria-encoded transcripts, including MT-CO1, MT-CO2, MT-ND3, and MT-ND4 mRNAs, suggesting that LANCL1-AS1 may functions coordinately with the RBP LRPPRC to maintain mitochondrial homeostasis, muscle contraction and thus affect muscle quality control and regeneration. ONGOING WORK AND FUTURE PLANS 1. To study the molecular mechanism whereby LANCL1-AS1 and LRPPRC regulate mitochondria functions. 2. To investigate LRPPRC and its downstream mitochondrial regulatory genes expression patterns in healthy human aging muscle (GESTALT). 3. To investigate the regulatory role of LANCL1-AS1 in mouse muscle regeneration in vivo.