[PhD Thesis Presentation_Zoom] ‐ Dong Cao – "Investigation of Circular RNA Regulation by Cis and Trans Elements in Caenorhabditis Elegans"

Circular RNAs (circRNAs) are regulatory molecules that show diverse functions. However, the regulation of circRNA formation is not well-understood. Through the large-scale isolation of neurons from the first larval stage of Caenorhabditis elegans, C. elegans neuronal circRNA profile was analyzed for the first time by RNA sequencing of the whole-transcriptome. Using circRNAs identified in this dataset, the regulation of circRNA by cis and trans elements was studied in vivo. It was found that reverse complementary matches (cis elements) found in introns that surround circRNA-forming exon(s) not only promote circRNA formation but also are involved in skipping of the exon(s). Through one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, it was uncovered that exon-skipping and back-splicing independently occur at the same time. Thirteen RNA binding proteins were screened as trans elements that regulate the formation of circRNAs in C. elegans. Among them, FUST-1, the homolog of FUS (fused in sarcoma), down-regulates both circRNA formation and exon skipping of the same pre-mRNA without affecting its cognate linear mRNA levels. In zip-2, the 5’ splice sites for back splicing and exon skipping seem to be important for FUST-1 to regulate exon-skipping and back-splicing, respectively. When two mutations (R446S and P447L), which are equivalent to natural mutations in the FUS nuclear localization signal (R524S and P525L) observed in amyotrophic lateral sclerosis, were introduced into FUST-1, both of the mutations dramatically down-regulated the formation of circRNAs. Moreover, an auto-regulation by FUST-1 "isoform a" was found to be required for the production of FUST-1 "isoform b", which has a different N-terminal sequence from "isoform a", by promoting the skipping of exon 5 of its own pre-mRNA. FUST-1, "isoform a" is a functional isoform for the regulation of circRNA formation, whereas "isoform b" is non-functional for the regulation exon-skipping or circRNA formation.