Kibiryeva, N. | Search bioRxiv

bioRxiv2021

microRNA Expression Levels Change in Neonatal Patients During and After Exposure to Cardiopulmonary Bypass

ObjectivesThe systemic inflammation that occurs after exposure to cardiopulmonary bypass (CPB), which is especially severe in neonatal patients, is associated with poorer outcomes and is not well understood. In order to gain deeper insight into how exposure to bypass activates inflammatory responses in circulating leukocytes, we studied changes in microRNA (miRNA) expression during and after exposure to bypass. miRNAs are small non-coding RNAs that have important roles in modulating protein levels and function of cells. MethodsWe performed miRNA-Sequencing on leukocytes isolated from neonatal cardiopulmonary bypass patients (N=5) at 7 timepoints during the process of CPB, including prior to the initiation of bypass, during bypass, and at three time points during the first 24 hours after weaning from bypass. We identified significant differentially expressed miRNAs using generalized linear regression models, and miRNAs were defined as statistically significant using an FDR adjusted p <0.05. We identified gene targets of these miRNAs using the Targetscan database, and identified significantly enriched biological pathways for these gene targets. ResultsWe identified 54 miRNAs with differential expression during and after CPB. These miRNAs clustered into 3 groups, including miRNAs that were increased during and after CPB (3 miRNAs), miRNAs that decreased during and after CPB (10 miRNAs), and miRNAs that decreased during CPB but then increased 8-24 hours after CPB. 38.9% of the target genes of these miRNAs were significantly differentially expressed in our previous study. miRNAs with altered expression levels are predicted to significantly modulate pathways related to inflammation and signal transduction. ConclusionsThe unbiased profiling of the miRNA changes that occur in the circulating leukocytes of bypass patients provides deeper insight into the mechanisms that underpin the systemic inflammatory response that occurs in patients after exposure to cardiopulmonary bypass. These data will help the development of novel treatments and biomarkers for bypass associated inflammation.

developmental biology↗

bioRxiv2019

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

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 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.\n\nAuthor 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.

genetics↗