PARP1 Regulates Circular RNA Biogenesis though Control of Transcriptional Dynamics

Abstract

Circular RNAs (circRNAs) are a recently discovered class of RNAs derived from protein-coding genes that have important biological and pathological roles. They are formed through backsplicing during co-transcriptional alternative splicing; however, the unified mechanism that accounts for backsplicing decisions remains unclear. Factors that regulate the transcriptional timing and spatial organization of pre-mRNA, including RNAPII kinetics, the availability of splicing factors, and features of gene architecture, have been shown to influence backsplicing decisions. Poly (ADP-ribose) polymerase I (PARP1) regulates alternative splicing through both its presence on chromatin as well as its PARylation activity. However, no studies have investigated PARP1’s possible role in regulating circRNA biogenesis. Here, we hypothesized that PARP1’s role in splicing extends to circRNA biogenesis. Our results identify many unique circRNAs in PARP1 depletion and PARylation-inhibited conditions compared to the wild type. We found that while all genes producing circRNAs share gene architecture features common to circRNA host genes, genes producing circRNAs in PARP1 knockdown conditions had longer upstream introns than downstream introns, whereas flanking introns in wild type host genes were symmetrical. Interestingly, we found that the behavior of PARP1 in regulating RNAPII pausing is distinct between these two classes of host genes. We conclude that the PARP1 pausing of RNAPII works within the context of gene architecture to regulate transcriptional kinetics, and therefore circRNA biogenesis. Furthermore, this regulation of PARP1 within host genes acts to fine tune their transcriptional output with implications in gene function.

Exploring the interplay between PARP1 and circRNA biogenesis and function

“…Given the effects of FUS and QKI in promoting back-splicing, it is not far-fetched to speculate that these RBPs cooperate with PARP1 to regulate circRNA expression during transcription. Finally, our recent finding shows that in the presence of PARP1 and/or PARylation, flanking introns of circRNAs are enriched in motifs recognized by these RPBs that are critical for circRNA biogenesis (Eleazer et al, 2023). Collectively, these studies provide supportive evidence that PARP1 and/or its PARylation activity adds to the complexity of circRNA processing pathways.…”

“…In detail, when exonic pausing is accompanied by an increase in intronic pausing, back-splicing events are more likely to occur, increasing the production of circRNAs and thereby the circRNA-to-linear mRNA ratio. However, when exonic pausing is coupled with decreased intronic pausing, linear RNA is favored (Eleazer et al, 2023). These results suggest that PARP1 is dynamically regulating RNAPII elongation in exons and introns, mediating the transcriptional output from a particular gene.…”

“…However, G-quadruplexes were depleted in host genes of circRNA producing genes and this depletion was exaggerated in PARP1 knockdown conditions. Lastly, differential RNAP binding motifs were seen in the presence and absence of PARP1, suggesting the possibility of PARP1 recruiting different RBPs to mediate circRNA biogenesis (Eleazer et al, 2023). In general, PARP1 seems to act together with cis-regulatory elements of the genome to mediate the production of different types of circRNAs.…”

“…In support of this idea, synthesis of long downstream introns favors back-splicing by increasing the elongation time of RNAPII along the gene body, making a downstream acceptor/linear splicing partner unavailable in comparison to an upstream donor/back-splicing partner (Santos-Rodriguez et al, 2021;Zhang et al, 2014). In support of PARP1's role for this process, we recently discovered that depletion of PARP1 resulted in the accumulation of RNAPII downstream of the back splice donor site, possibly extending the time required for the linear acceptor to be produced to facilitate back-splicing (Eleazer et al, 2023). While our research is beginning to elucidate the role of PARP1 in circRNA production, more studies are needed to investigate all conceivable pathways by which PARP1 mediates the lariat-driven circularization model.…”

“…Such mechanisms aid in co-transcriptional splicing. We recently showed that PARP1-chromatin state regulates pausing at these boundaries to enact co-transcriptional splicing decisions (Eleazer et al, 2023;Gibson et al, 2016;Matveeva, Al-Tinawi, et al, 2019). Recent studies have shown that circRNA expression is mediated not only post-transcriptionally (Zhang et al, 2016), but also co-transcriptionally and that splicing is the primary regulatory step in transcript circularization (Ashwal-Fluss et al, 2014).…”