Coilin enhances phosphorylation and stability of DGCR8 and promotes miRNA biogenesis

Abstract

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is comprised of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal Body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of co-immunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.

Coilin mediates m6A RNA methylation through phosphorylation of METTL3

“…For the meRIP assay, our qRT-PCR targets were pri-Let7a and pri-miR-21. Pri-Let7a is a miRNA whose biogenesis our lab has found to be regulated by coilin ( Logan et al, 2020 ; Lett et al, 2021 ) and pri-miR-21 is a novel miRNA whose coilin-mediated biogenesis is being investigated for the first time. Both miRNAs have been previously described as being regulated by m 6 A modification ( Konno et al, 2019 ; Ye et al, 2022 ).…”

“…In wanting to understand how coilin might participate in miRNA biogenesis, our lab has investigated coilin's interaction with the microprocessor complex. To summarize, we have found that coilin suppression reduces DGCR8 phosphorylation and SUMOylation, resulting in decreased protein stability and blunting of microprocessor activity ( Lett et al, 2021 , 2023 ).…”

“…Our lab has also uncovered a role for the Cajal body (CB) marker protein coilin as a positive regulator of miRNA biogenesis ( Logan et al, 2021 , 2022 , 2020 ; Lett et al, 2021 ). Canonically, coilin function has been largely associated with its CB marker role, wherein coilin maintains CB assembly ( Bohmann et al, 1995 ; Hebert and Matera, 2000 ; Hebert et al, 2002 ) and spliceosome efficiency ( Bellini and Gall, 1998 ; Sleeman et al, 2001 ; Tucker et al, 2001 ).…”

Coilin as a regulator of NF-kB mediated inflammation in preeclampsia

“…We have previously published that the reduction of proteins enriched in the CB decreases miRNA biogenesis ( Lett et al, 2021 ; Logan et al, 2021 , 2020 ). Importantly, we have also published that coilin reduction in primary cell lines that have few CBs, such as WI-38, also results in decreased miRNA levels, suggesting that coilin contributes to miRNA biogenesis in cells with or without CBs ( Lett et al, 2021 ; Logan et al, 2021 , 2020 ). Therefore, we hypothesize CBs could be an upstream regulator of the miR-517-3p pathway that involves NF-κB and leads to the upregulation of sFlt-1 in hypoxia ( Fig.…”

“…We have previously identified the Cajal bodies (CBs) marker protein, coilin, as a novel regulator of miRNA biogenesis ( Lett et al, 2021 ; Logan et al, 2021 , 2020 ). CBs are phase-separated nuclear condensates that contribute to the biogenesis and maturation of ribonucleoproteins (RNPs).…”