Nagasawa Chloe K, Kibiryeva Nataliya, Marshall Jennifer, O'Brien James E, Bittel Douglas C
Kansas City University of Medicine and Biosciences, Kansas City, MO, USA.
Ward Family Heart Center, Children's Mercy Hospital, Kansas City, MO, USA.
Pediatr Cardiol. 2020 Feb;41(2):341-349. doi: 10.1007/s00246-019-02263-4. Epub 2020 Jan 17.
The heart is the first major organ to develop during embryogenesis and must receive proper spatiotemporal signaling for proper development. Failure of proper signaling between the first and second heart fields at twenty days gestation contributes to the generation of a congenital heart defect. The most common cyanotic congenital heart defect is tetralogy of Fallot (TOF) which requires surgical intervention in the first year of life. In right ventricular tissue of infants born with TOF, the levels of scaRNA1 are reduced and mRNA splicing is dysregulated. In this study, we investigate a method of quantifying pseudouridylation levels in relation to scaRNA1 levels in spliceosomal RNA U2 in three different groups of samples: right ventricular (RV) tissue of infants born with TOF versus RV tissue from normally developing infants, scaRNA1 knockdown in primary normal cardiomyocytes derived from normally developing infants, and scaRNA1 overexpression in primary cells derived from RV tissue from infants born with TOF. We hypothesize that the amount of pseudouridylation is dependent on scaRNA1 level, compromising spliceosomal function and therefore, contributing to the generation of a congenital heart defect. Our results revealed a statistically significant decrease of pseudouridylation levels in the right ventricular tissue of infants born with TOF compared to the controls. Knocking down the scaRNA1 levels in normal primary cardiomyocytes resulted in a statistically significant decrease of pseudouridylation. Finally, an overexpression of scaRNA1 in TOF primary cells resulted in an increase in pseudouridylation levels, but it did not achieve statistical significance. Our previous research provided an association between scaRNA levels, alternative splicing, and development. Here, we demonstrate that pseudouridylation levels in spliceosomal RNA U2 is dependent on the expression level of scaRNA1. Although further investigation is needed, we believe that scaRNA expression regulates biochemical modifications to spliceosomal RNAs, adjusting the fidelity of the spliceosome, allowing for controlled alternative splicing of mRNA that is important in embryonic development. If validated, this is an underappreciated mechanism that is critical for regulating proper embryonic development.
心脏是胚胎发育过程中第一个发育的主要器官，必须接受适当的时空信号才能正常发育。妊娠20天时，第一和第二心脏区域之间的信号传递出现异常会导致先天性心脏缺陷。最常见的青紫型先天性心脏缺陷是法洛四联症（TOF），需要在出生后第一年进行手术干预。在患有TOF的婴儿的右心室组织中，scaRNA1水平降低，mRNA剪接失调。在本研究中，我们研究了一种量化假尿苷化水平的方法，该方法与三组不同样本中剪接体RNA U2中的scaRNA1水平相关：患有TOF的婴儿的右心室（RV）组织与正常发育婴儿的RV组织、来自正常发育婴儿的原代正常心肌细胞中的scaRNA1敲低，以及来自患有TOF的婴儿的RV组织的原代细胞中的scaRNA1过表达。我们假设假尿苷化的量取决于scaRNA1水平，损害剪接体功能，因此导致先天性心脏缺陷的产生。我们的结果显示，与对照组相比，患有TOF的婴儿的右心室组织中的假尿苷化水平在统计学上显著降低。在正常原代心肌细胞中敲低scaRNA1水平导致假尿苷化在统计学上显著降低。最后，在TOF原代细胞中过表达scaRNA1导致假尿苷化水平增加，但未达到统计学显著性。我们之前的研究提供了scaRNA水平、可变剪接和发育之间的关联。在这里，我们证明剪接体RNA U2中的假尿苷化水平取决于scaRNA1的表达水平。尽管需要进一步研究，但我们认为scaRNA表达调节剪接体RNA的生化修饰，调整剪接体的保真度，允许对胚胎发育中重要的mRNA进行可控的可变剪接。如果得到验证，这是一种未被充分认识的机制，对调节正常胚胎发育至关重要。
