Defining an evolutionarily conserved role of GW182 in circular RNA degradation

Abstract

Dear Editor, Circular RNAs (circRNAs) are covalently closed RNA molecules derived from thousands of protein-coding genes via “backsplicing”. In many cases, the “backsplicing” step can be trigged by the flanking complementary intronic repeat elements that efficiently bring the intervening splicing sites into close proximity. Most circRNAs are cytoplasmic, and nuclear export of mature circRNAs is regulated in a length-dependent manner. While the functions of circRNAs remain largely unknown, recent reports have revealed that some circRNAs can control gene expression by affecting transcription, acting as splicing regulators and mircoRNA sponges. It is also becoming evident that circRNAs are associated with several diseases such as cancer and brain disorders. Due to the lack of a defined 5′ or 3′ end, circRNAs are naturally more stable than their parental linear RNAs as they are not targets for the exosome or exonuclease. This was exemplified by the circRNAs derived from Drosophila dati and laccase2 gene or our previously described expression plasmids (Fig. 1a; Supplementary Fig. S1). Nevertheless, how circRNAs are degraded or what factors contribute to a surveillance pathway is largely unclear. To reveal the factors which are required for degradation of circRNAs, we employed a focused RNAi screening in Drosophila DL1 or S2 cells targeting 31 genes with known roles in RNA metabolism, and then assessed the levels of steady-state circRNAs by qRT-PCR (Fig. 1b; Supplementary Figs. 2–3. In line with previous study, knockdown of RNA decay factors Pop2 (also known as CAF1), Not1, and DCP2 led to significant accumulation of Vha68-1 mRNA (Supplementary Fig. S2b), but had no effect on the levels of steady-state circRNAs (Fig. 1b). We instead found that depletion of GW182 resulted in accumulation of both steady-state circdati and circlaccase2 transcripts (Fig. 1b). GW182 is a key component of P-body and RNAi machine as it facilitates the assembly of P-body and acts as a molecular scaffold bringing together RNA-induced silencing complexes and various mRNA decay enzymes. However, depletion of other P-body components or RNAi machine factors did not have effect on steady-state circRNA levels, indicating that P-body or RNAi machine does not affect circdati or circlaccase2 degradation (Fig. 1b). To examine whether GW182 exerts a general or limited role in controlling circRNA levels, we tested the levels of 12 additional circRNAs that were of varying length and exon counts in GW182-depleted DL1 cells (Fig. 1c; Supplementary Table S1). The levels of most steady-state circRNAs were significantly increased upon GW182 depletion. Importantly, the role of GW182 appears to be robust in affecting circRNA stability because (i) the levels of most nascent circRNAs were not affected upon GW182 depletion, suggesting that circRNA biogenesis is largely unaffected by GW182 (Fig. 1d; Supplementary Fig. S4a, b), (ii) circRNA accumulation was also observed in GW182depleted Drosophila S2 cells genome widely (Fig. 1e–g; Supplementary Figs. S4–S6), (iii) the enriched circRNAs were also verified using a secondary GW182 dsRNA (Supplementary Fig. S4c, d), (iv) overexpression of GW182 decreased the levels of steady-state circRNAs (Supplementary Fig. S4e), (v) depletion of GW182 had no effect on nuclear circRNA levels, while cytoplasmic circRNAs accumulated (Fig. 1h; Supplementary Fig. S7), and (vi) GW182 depletion had little effect on degradation of circRNAs’ parental mRNAs (Fig. 1f, g; Supplementary Figs. S4d, S6c–e). Taken together, these data suggested