Abstract 378: Physiologic Role of Rpl13a SnoRNAs in Cell Size Regulation

Objectives: Box C/D small nucleolar RNAs (snoRNA) are a multifunctional family of ncRNAs that play a critical role in guiding 2'- O -methylation (Nm) of ribosomal RNA (rRNA). My work has recently revealed that box C/D snoRNAs can also direct methylation of messenger RNA (mRNA), and that this methylation can regulate mRNA translation in the heart. In studies of genetically-engineered mice lacking four box C/D snoRNAs, I have observed that the knockout animals have relatively small hearts compared to wild-type. The objective of my current study is to define the specific mechanisms by which box C/D snoRNAs regulate heart size. Methods: I investigated the hearts of genetically-engineered mice lacking four box C/D snoRNAs from the Rpl13a locus (snoRNAs U32a, U33, U34 and U35a ). I also used antisense oligonucleotides to knock down these snoRNAs in H9c2 rat cardiomyoblasts. Changes in gene and protein expression were assessed by qPCR and immunoblot. Relative Nm modification of mRNA was determined by reverse transcription at low dNTP concentrations, followed by real-time PCR (RTL-P). Results: Germline knockout of the four box C/D Rpl13a snoRNAs ( U32a, U33, U34 and U35a ) in adolescent mice produces developmentally smaller hearts. In H9c2 rat cardiomyoblasts, knockdown of Rpl13a snoRNAs by antisense oligonucleotides significantly reduces H9c2 cell size. These concordant effects on organ and cell size suggest that the Rpl13a snoRNAs might be regulating critical pathways that determine cell growth. Using a candidate gene approach, I found that Mtor mRNA and protein expression is significantly reduced in hearts from Rpl13a snoRNA knockout mice. Preliminary results using the RTL-P method suggest that Mtor is subject to snoRNA-guided Nm modification, which is decreased in Rpl13a snoRNA knockout mouse hearts. Conclusion: These results suggest that Rpl13a snoRNAs regulate cardiomyocyte growth, at least in part, by guiding Nm modification of Mtor mRNA.