Snord94 expression level alters methylation at C62 in snRNA U6

Abstract

Understanding 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 incomplete. Notably, a subset of small noncoding RNAs known as scaRNAs (small cajal body associated RNAs) contribute to spliceosome maturation and function through guiding covalent modification of spliceosomal RNAs with either methylation or pseudouridylation on 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 guides methylation on one specific cytosine (C62) on spliceosomal RNA U6, thus potentially altering spliceosome function during embryogenesis. We previously showed that in the myocardium of infants with heart defects, mRNA is alternatively spliced as compared to control tissues. We also demonstrated that alternatively spliced genes were concentrated in the pathways that control heart development. 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 its target region in U6, suggesting a potential mechanism for modifying alternative splicing of mRNA. The potential importance of scaRNAs as a developmentally important regulatory mechanism controlling alternative splicing of mRNA is unappreciated and needs more research.

The Interlocking Lives of LARP7: Fine-Tuning Transcription, RNA Modification, and Splicing through Multiple Non-coding RNAs

“…Alternatively, the loss of 2 0 -O-methylations might affect unwinding kinetics of the U4/U6 duplex during spliceosome activation as many of the modifications map to the U4/U6 stem II (Figure 1B, left). Consistently with present studies, loss of an individual U6 2 0 -O-methylation has been linked to defects or alterations in splicing in a subset of introns (Ogren et al, 2019). The nature of how the possible structural perturbations translate into the observed changes in splicing needs further investigation.…”

A contemporary review of snoRNAs in cardiovascular health: RNA modification and beyond

“… 41 When analyzing the biopsied umbilical arterial fibroblast cells, the expression of SNORD113-6 was much higher than controls. 41 More specifically, SNORD113-6 and the murine equivalent, AF357425, were believed to methylate tRNA Leu (TAA) and prevent the fragmentation of its original structure into smaller tRFs, based on the high-to-low ratio of tRNA Leu (TAA) to tRF Leu 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ( Figure 2 ). 41

Figure 2 The breakdown of tRNA in a eukaryotic cell When cells are exposed to stress hypoxia, tRNA molecules degrade into tRFs, molecules that can be detrimental to cellular health (bottom row).

…”

“… 50 Another study demonstrated that SNORD94, a specific scaRNA, guides Cm62 in U6 snRNA and alters overall spliceosome function. 51 Whether scaRNA expression in TOF can be mechanistically induced to alleviate TOF symptoms deserves further attention.

Figure 3 Hearts of infants with TOF can have an abnormal hole located between the right and left ventricles, resulting in the pumping of deoxygenated blood through the aorta and into the body In the affected heart, it is believed that the underexpression of scaRNA1, SNORD94, and possibly other undiscovered sno-/scaRNAs may disrupt several molecular processes, such as splicing, transcription, and translation, as well as alter cellular products, such as spliceosomes, mRNA, rRNA, ribosomes, and proteins.

…”

“… 41 SNORD94 Human, 2, 2p11.2 Congenital heart defects Upregulated Targets a cytosine on spliceosomal RNA subunit U6 via Nm Human infant right ventricle tissue, primary neonatal cardiomyocytes Ogren et al. 51 SNORD96A, SNORD73A Human, 5, 5q35 and 4, 4q31, respectively Hypertrophic cardiomyopathy Upregulated Regulate alternative splicing via Nm hi-PSC-CMs James et al. 55 …”

Small but strong: the emerging role of small nucleolar RNA in cardiovascular diseases

“…These observations strongly suggest that snoRNAs and snRNAs are necessary for normal cardiac development. Furthermore, Ogren et al reported that methylation in the target region of SNORD94 on U6 is decreased in right ventricular myocardium tissue of infants with TOF compared with that of the control ( Ogren et al, 2019 ). Additionally, cell culture experiments revealed that, by changing the levels of SNORD94, a corresponding change in methylation at C 62 within the snRNA U6 is produced.…”