STRIATED MUSCLE SPECIFIC RIBOSOMAL PROTEIN L3-LIKE: EFFECT OF KNOCKOUT ON CARDIAC FUNCTION AND PROTEIN TRANSLATION

Ribosomes are the molecular machinery of the cell that catalyzes synthesis of peptides from amino acids. The eukaryotic ribosome is made up of four strands of ribosomal RNA (rRNA) and ~80 ribosomal proteins. While many tissues routinely exhibit variations of ribosomal protein stoichiometry, tissue specific ribosomal proteins are rare. The ribosomal protein with the highest tissue specificity of any ribosomal protein is found in striated muscle, ribosomal protein L3-like (RPL3L). Other than its tissue specificity, association with atrial fibrillation, and chromosomal location, there is little known about the function of RPL3L. However, its ubiquitously expressed paralog, RPL3, has been well documented to be essential for ribosome biogenesis, aid in peptidyl transfer, and increase translational fidelity. This thesis, therefore, seeks to address the critical gap in knowledge on the function of RPL3L in striated muscle and specifically, the effect of RPL3L knockout (KO) on cardiac function and protein translation in vivo. To that end, a RPL3L KO mouse was generated that, instead of striated muscle-specific RPL3L, expresses the ubiquitous RPL3 in striated muscles. The first aim of this dissertation was to test the hypothesis that RPL3L KO would induce cardiac arrhythmias by expression in the atria. First the expression pattern of RPL3 and RPL3L in the wild-type (WT) heart were established by both RT-PCR and Western blot. Both indicated that while the ventricle has high expression of RPL3L, RPL3 is found at much lower levels (~10% that of RPL3L). The atria however, had the opposite expression pattern with RPL3 being high and RPL3L not expressed. In order to determine if the RPL3L KO mice recapitulated the fibrillation phenotype seen in humans with Rpl3l variants, we performed echocardiography and electrocardiography on WT and KO mice. No changes were observed in heart rate, ejection fraction, wall thickness during systole or diastole, fractional shortening or stroke volume under resting conditions. When telemetry fitted mice were treated with the β2 adrenergic receptor agonist, isoproterenol, both WT and KO mice showed a significant increase in heart rate after treatment (p=0.02 and 0.0007 respectively) but the rate of response was significantly more rapid in KO mice (p= \u3c 0.0001). Due to the increase in rate of response to isoproterenol in the KO, we hypothesized that loss of expression of RPL3L in the pace-making center of the heart, the sinoatrial node, was responsible for the rapid increase in heart rate To that end, single-cell RNA sequencing data from nuclei of the sinoatrial node, and proteomic data from the sinoatrial node were queried. Analysis revealed that RPL3L is expressed at a very low level at the mRNA level in the sinoatrial node but that it is not detected at the protein level. These results do not support the hypothesis that loss of RPL3L in the atria causes atrial fibrillation, rather this evidence suggests that if RPL3L plays a role in atrial fibrillation, it is likely secondary to a ventricular pathology. The second aim of this dissertation was to test the hypothesis that RPL3L plays a functionally specialized role in the ribosome causing enhanced translation of a subset of mRNAs, thereby conferring preferential recruitment to mRNAs which are specific to striated muscle. Actively translating ribosomes of cardiac tissue were isolated via polysome fractionation and were subsequently subjected to RNA sequencing (RNA-seq). Analysis revealed that there were 216 mRNAs that were differentially translated (but not differentially transcribed). Of these mRNAs, 68 were more highly translated in WT (RPL3L-ribosomes) whereas 148 were more highly translated in the KO (RPL3-conatining ribosomes). Gene ontology of differentially translated mRNAs showed highest enrichment for genes involved in RNA binding and splicing. These results support the hypothesis that there is differential translation of a subset of mRNAs This study demonstrates that KO of RPL3L is not lethal, and while it does cause changes in cardiac response to isoproterenol, its loss is not sufficient to induce atrial fibrillation in mice. This study also demonstrates that RPL3L expression is robust and highly specific to the ventricles of the heart but that its expression exhibits only minor alterations on the cardiac translatome. The findings here help to further our understanding of translation in the heart and its effects on cardiac physiology