A Tale of RNA Circles: Function of Circular RNAs in Reproduction
Biochemistry and Molecular Biology
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CircRNAs are a group of RNA circles harboring exons out of order from their normal genomic contexts. Although discovered ~20 years ago, circRNAs have since then been overlooked because they were regarded as “transcriptional noises” without specific functions. CircRNAs have returned to the spotlight recently because a large number of circRNAs have been identified to display tissue- or developmental stage-specific expression patterns and many appear to have regulatory roles. In this dissertation, I first briefly reviewed the history of circRNA study and the latest major findings regarding circRNA functions (Chapter I). Soon after we started working on circRNAs, we realized that almost all currently available circRNA discovery and detection methodologies use MMLV-derived reverse transcriptase (e.g., SuperScript II), which we found produces artificial junction sequences and thus, leads to false identification of circRNAs. To overcome the problem, we embarked on developing better approaches for identification and detection of true RNA circles (Chapter II). CircRNAs are more abundant in brain and testis than in other organs, suggesting they may have important neuronal function and regulate spermatogenesis. Indeed, brain circRNAs are significantly upregulated with aging. Through comprehensive transcriptomic and epi-transcriptomic analyses, we discovered that circRNA production increases while their linear forms actively undergo degradation in developing male germ cells; circRNAs tend to form between m6A-enriched sites on mRNAs, which are usually located around the start and stop codons. Consequently, ~46% of these spermiogenesis-enriched circRNAs contain translatable CDS with an m6A-modified start codon located within their junction sites. The dynamic spatiotemporal production and subcytoplasmic compartmentalization, as well as the delicate regulation by m6A writer METLL3 and eraser ALKBH5, all suggest that of these CDS-containing circRNAs function to protect certain mRNAs from massive degradation in the elongation stage of spermiogenesis (Chapter III). These data uncovered not only a novel role of m6A in generating circRNAs with coding potential, but also an alternative mechanism for delayed translation which is required for protein synthesis during spermiogenesis.