Dataset Information

Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity.

ABSTRACT: The sequence and roles in developmental progression of the microRNA let-7 are conserved. In general, transcription of the let-7 primary transcript (pri-let-7) occurs early in development, whereas processing of the mature let-7 microRNA arises during cellular differentiation. In Caenorhabditiselegans and other animals, the RNA-binding protein LIN-28 post-transcriptionally inhibits let-7 biogenesis at early developmental stages, but the mechanisms by which LIN-28 does this are not fully understood. Nor is it understood how the developmental regulation of let-7 might influence the expression or activities of other microRNAs of the same seed family. Here, we show that pri-let-7 is trans-spliced to the SL1 splice leader downstream of the let-7 precursor stem-loop, which produces a short polyadenylated downstream mRNA, and that this trans-splicing event negatively impacts the biogenesis of mature let-7 microRNA in cis Moreover, this trans-spliced mRNA contains sequences that are complementary to multiple members of the let-7 seed family (let-7fam) and negatively regulates let-7fam function in trans Thus, this study provides evidence for a mechanism by which splicing of a microRNA primary transcript can negatively regulate said microRNA in cis as well as other microRNAs in trans.

SUBMITTER: Nelson C

PROVIDER: S-EPMC6432665 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity.

Nelson Charles C Ambros Victor V

Development (Cambridge, England) 20190304 5

The sequence and roles in developmental progression of the microRNA let-7 are conserved. In general, transcription of the let-7 primary transcript (pri-let-7) occurs early in development, whereas processing of the mature let-7 microRNA arises during cellular differentiation. In Caenorhabditiselegans and other animals, the RNA-binding protein LIN-28 post-transcriptionally inhibits let-7 biogenesis at early developmental stages, but the mechanisms b ...[more]

PMID: 30770392

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Project description:Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3β, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

Dataset Information

Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity.

Publications

Trans-splicing of the <i>C. elegans let-7</i> primary transcript developmentally regulates <i>let-7</i> microRNA biogenesis and <i>let-7</i> family microRNA activity.

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