Abstract P1111: Dysfunctional SCARNAs And Failed Pseudouridylation Result In Irregulation Of Cardiac Genes In Noonan Syndrome Patient-derived Induced Cardiomyocytes

Abstract

Background: Small Cajal body associated RNAs (scaRNAs) can control alternative splicing and the maturation of mRNAs by regulating the pseudouridylation (PsU) of small nuclear RNAs (snRNAs), which are involved in the formation of the spliceosome complex, via RNA-dependent mechanism. Failure of snRNA PsU results in dysregulated mRNA splicing and maturation which causes congenital heart defects. In this study, we aim to find the importance of snRNAs PsU in Noonan syndrome (NS) patients-derived induced pluripotent stem cell-derived cardiomyocytes (NS-iCM) when compared to normal skin fibroblast iCM (SF-iCM). Hypothesis: We hypothesize that snRNAs PsU by scaRNAs will have a significant role in the development of cardiomyocytes in NS patients. Methods and Results: We generated iCMs from SF-iPSCs and NS patient-derived NS-iPSCs. The qRT-PCR analysis showed a significantly lower expression of scaRNA6, scaRNA20, and scaRNA28 in NS-iCMs when compared with control SF-iCMs (Fig. A). The PsU of snRNA U12 was quantified by RNA Immunoprecipitation (RIP) using the Pseudouridine (Ψ)-specific antibody and mouse IgGas a negative control. The data showed that snRNA U12 was significantly reduced in the NS-iCM when compared with negative IgG (Fig. B). Furthermore, the PsU modification was quantified by using the 1-cyclohexyl- (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) method. Our data showed that relative Ψ units were significantly reduced in the NS-iCMs when compared with normal SF-iCMs (Fig. C). Conclusion: Our results indicate that snRNAs PsU by scaRNAs were dysfunctional in NS-iCMCs, which may result in cardiac defects in NS patients.