Methylation at nucleotide C62 in spliceosomal RNA U6 alters mRNA splicing which is important for embryonic development

Abstract

AbstractUnderstanding the regulation of development can help elucidate the pathogenesis behind many developmental defects found in humans and other vertebrates. Evidence has shown that alternative splicing of messenger RNA (mRNA) plays a role in developmental regulation, but our knowledge of the underlying mechanisms that regulate alternative splicing are inadequate. Notably, a subset of small noncoding RNAs known as scaRNAs (small cajal body associated RNAs) contribute to spliceosome maturation and function through covalently modifying spliceosomal RNAs by either methylating or pseudouridylating specific nucleotides, but the developmental significance of these modifications is not well understood. Our focus is on one such scaRNA, known as SNORD94 or U94, that methylates one specific cytosine (C62) on spliceosomal RNA U6, thus potentially altering spliceosome function during embryogenesis. We previously showed that mRNA splicing is significantly different in myocardium from infants with congenital heart defects (CHD) compared to controls. Furthermore, we showed that modifying expression of scaRNAs alters mRNA splicing in human cells, and zebrafish embryos. Here we present evidence that SNORD94 levels directly influence levels of methylation at C62 in U6, which we have previously shown is associated with altered splicing and congenital heart defects. The potential importance of scaRNAs as a developmentally important regulatory mechanism controlling alternative splicing of mRNA is unappreciated and needs more research.Author summarySplicing of mRNA transcripts by removal of introns and some non-critical exons is a crucial part of mRNA processing, gene expression, and cell function, and regulation of this process is still under investigation. Alternative splicing of mRNA transcripts of genes is tissue and time specific throughout life, although this process occurs everywhere in the body according to local tissue needs and signals. The spliceosome, the large ribonucleoprotein complex that carries out splicing, is biochemically modified by small noncoding RNAs, which is important for its structure and function. Here we show that the amount of 2’-O-ribose methylation at nucleotide C62 in spliceosomal RNA U6 is dependent on the level of the scaRNA SNORD94. We hypothesize that alternative splicing is dependent, at least in part, on biochemical modification to the spliceosomal RNAs. Furthermore, when scaRNA directed modifications are dysregulated, the result causes inappropriate alternative splicing that may contribute to developmental defects such as congenital heart defects. To our knowledge, this is the first demonstration that 2’-O-ribose methylation is indeed dependent on scaRNA levels in human cells and tissues.