Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene

Abstract

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 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 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 ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.

Beyond Back Splicing, a Still Poorly Explored World: Non-Canonical Circular RNAs

“…Just because the two boundaries identified in the circular junction sequence are mapped in intronic sequences does not necessarily imply a lariat-derived intronic circRNA. We reviewed several published lists in which we expected to find lariat-derived intronic circRNAs and were disappointed, except for a very recent article by Das et al published in 2020 [ 24 ]. In the literature, we can easily find articles that compare computational tools dedicated to circRNA identification [ 26 , 27 ] and we note that they correctly report the consideration of circRNAs when originating from back splicing.…”

“…In our opinion, with this filter, it is highly unlikely that a lariat-derived intronic circRNA will be found among the circRNAs detected, even though they may be classified by authors as ‘intronic’. We greatly appreciated to note [ 24 ] that CIRCexplorer2 [ 34 ] considers two models of circRNA (exonic and lariat-derived intronic). In addition to tools developed to detect “all circRNAs”, we would like to mention a tool designed to work on intronic reads [ 35 ] and the development of a dedicated approach to characterize intron-derived circRNAs [ 20 , 21 ].…”

“…(4) A recent study of neuronal cells (neuronal projections and synapses) in mice identified 278 introns as able to produce sisRNA, and reads containing circular junctions were detected in 14 of them [ 22 ]. (5) Das et al [ 24 ] identified circRNAs in mouse pancreatic β-cells and highlighted several lariat-derived circRNAs originating from Ins2 .…”

“…Thanks to studies on human branch points, we know a distance of 9–400 bp between the branch point and the 3′ end of the intron is possible [ 37 ], even though 90% of branch points occur within 19 to 37 nucleotides upstream of the 3′ splice site [ 16 ]. After examining published lariat-derived circRNAs [ 20 , 21 , 23 , 24 ], we have no objection considering a distance between the branch point and the 3′ end of the intron ranging from 12 to 60 nt for lariat-derived circRNAs. Moreover, when examining the sequence of reads spanning the circular junction of a lariat derived intronic circRNA, heterogeneity can sometimes be observed, as if several branch points are possible [ 20 , 21 , 24 ].…”

“…To become circular sisRNA, the lariat escapes the action of the debranching enzyme ( Figure 1 ). Several studies showed that lariat-derived circRNAs originate from introns that do not use an adenosine at the branch point but rather a ‘C’, or more rarely, a ‘G’ or ‘T’ [ 6 , 20 , 21 , 22 , 23 , 24 , 25 ]. The lariat debranching enzyme is hypothesized to be incapable of hydrolyzing the 2′−5′ bond at these residues [ 15 ].…”