The rRNA epitranscriptome and myonuclear SNORD landscape in skeletal muscle fibers contributes to ribosome heterogeneity and is altered by hypertrophic stimulus

In cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent post-transcriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'-O-Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'-O-Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq. We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1) induces decreased 2'-O-Me at a subset of skeletal muscle rRNAand 2) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'-O-Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'-O-Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes. Experiment 1: Whole skeletal muscle RNA following 3, 7, and 14 days of mechanical overload (MOV) and a sham surgery control condition in C57BL/6J (females, 5 months of age, n=12) were used for RiboMeth-seq analysis. MOV was induced by synergist ablation for which a small portion of the gastrocnemius muscle was removed (22), while sham surgery involved the same steps of synergist ablation without the removal of muscle. Experiment 3: A skeletal muscle specific RiboTag mouse (HSA-RiboTag) was generated by crossing the HSA-mER-CRE-mER (MCM) mouse (Jax, Stock No. 025750) with the RiboTag mouse (Jax, Stock No. 029977), which enabled cre-mediated hemagglutinin (HA) epitope tagging of ribosomes at the ribosomal protein L22 locus in a skeletal muscle-specific manner. HSA-RiboTag mice (females, 4-6 months of age, n=3 overload/sham) were administered tamoxifen (75 mg /kg body weight, corn oil solution) by intraperitoneal injection for five days starting on the day of MOV. For this experiment, MOV was performed on one limb with the contralateral limb serving as sham control.