Polo-like激酶1(PLK1),细胞周期进程的关键介质,与不良预后相关,并且是许多恶性肿瘤的治疗靶标。已经在Drosha上鉴定了PLK1的推定磷酸化位点,负责miR生物发生的微处理器的主要催化成分。几种激酶,包括GSK3β,p70S6激酶,ABL,PAK5,p38MAPK,CSNK1A1和ANKRD52-PPP6C,已被证明可以磷酸化miR生物发生机制的成分,改变他们的活动和/或本地化,因此不同miR亚群的生物发生。我们假设PLK1通过Drosha磷酸化调节miR生物发生。体外激酶测定证实了在S300和/或S302处Drosha的PLK1磷酸化。PLK1抑制降低了Drosha的丝氨酸磷酸化水平及其与DGCR8的RNA依赖性关联。相比之下,“磷酸模拟物”Drosha突变体显示与DGCR8的相关性增加。Drosha的PLK1磷酸化改变了Drosha微处理器复杂的亚细胞定位,由于PLK1抑制增加了DGCR8和Drosha的细胞溶质蛋白水平,而细胞核水平下降。重要的是,上述效应独立于PLK1的细胞周期调节作用,由于Drosha改变:PLK1抑制后的DGCR8定位发生在M期细胞显著积累之前,在M期停滞细胞中,PLK1调节的miRs没有增加。小RNA测序和qPCR验证用于评估PLK1活性对miR生物发生的下游后果,鉴定一组十个miR(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和miR-892a)的表达水平在统计学上被两种药理学PLK1激酶结构域抑制剂下调,RO-5203280和GSK461364。相反,转染野生型或组成型活性PLK1后,观察到这些miR水平升高。重要的是,pre-miR水平在PLK1抑制后降低,和pri-miR水平在PLK1激活后降低,因此,PLK1Drosha磷酸化在pri-miR-to-pre-miR加工水平调节miR生物发生。结合之前的研究,这项工作将DroshaS300和S302确定为几种激酶信号的主要整合点,其相对活性将决定不同miR亚群的相对生物发生效率。鉴定的激酶调节miR具有用作激酶抑制剂反应预测生物标志物的潜力,癌症和其他疾病。
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.