quantification-of-circular-rnas-using-digital-droplet-pcr

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Institute of Life Sciences

KIIT University

Skeletal muscles have an immense ability to regenerate from the muscle stem cells called satellite cells. The process of skeletal muscle regeneration is called myogenesis, which starts with activation of quiescent satellite cells immediately after muscle injury followed by proliferation and fusion of myoblasts into myotubes. Myogenesis is orchestrated through the expression of a specific set of genes which, at each step regulated by complex gene regulatory networks. Besides the well-established roles of transcription factors, increasing evidence demonstrated that circular (circ)RNAs modulate gene expression during myogenesis and are involved in muscle-related diseases. Here we review the recent findings of circRNAs involved in myogenesis.

Circular RNAs in myogenesis.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic β-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 () gene and are termed as circular intronic (). The expression of  transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of  was altered in βTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with  and their flanking region, suggesting their role in the  function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with . Gene ontology and pathway analysis of genes targeted by miRNAs associated with  suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially  transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Circular RNAs (circRNAs) are emerging as novel regulators of gene expression in various biological processes. CircRNAs regulate gene expression by interacting with cellular regulators such as microRNAs and RNA binding proteins (RBPs) to regulate downstream gene expression. The accumulation of high-throughput RNA-protein interaction data revealed the interaction of RBPs with the coding and noncoding RNAs, including recently discovered circRNAs. RBPs are a large family of proteins known to play a critical role in gene expression by modulating RNA splicing, nuclear export, mRNA stability, localization, and translation. However, the interaction of RBPs with circRNAs and their implications on circRNA biogenesis and function has been emerging in the last few years. Recent studies suggest that circRNA interaction with target proteins modulates the interaction of the protein with downstream target mRNAs or proteins. This review outlines the emerging mechanisms of circRNA-protein interactions and their functional role in cell physiology.

Emerging Role of Circular RNA-Protein Interactions.

Circular RNAs (circRNAs) are a newly discovered family of regulatory RNAs generated through backsplicing. Genome-wide profiling of circRNAs found that circRNAs are ubiquitously expressed and regulate gene expression by acting as a sponge for RNA-binding proteins (RBPs) and microRNAs (miRNAs). To identify circRNAs expressed in mouse skeletal muscle, we performed high-throughput RNA-sequencing of circRNA-enriched gastrocnemius muscle RNA samples, which identified more than 1,200 circRNAs. In addition, we have identified more than 14,000 and 15,000 circRNAs in aging human skeletal muscle tissue and satellite cells, respectively. A subset of abundant circRNAs was analyzed by RT-PCR, Sanger sequencing, and RNase R digestion assays to validate their expression in mouse skeletal muscle tissues. Analysis of the circRNA-miRNA-mRNA regulatory network revealed that conserved  might associate with miR-204-5p, a suppressor of myocyte enhancer factor 2c () expression. To support the hypothesis that  might regulate myogenesis by controlling  expression, silencing  moderately reduced  mRNA expression and inhibited C2C12 differentiation. We propose that  promotes  expression during muscle cell differentiation in part by acting as a sponge for miR-204-5p.

Arundhati Das

Institute of Life Sciences

Quantification of Circular RNAs Using Digital Droplet PCR