Regulation of the microRNA Induced Silencing Complex in C. elegans.

[摘要] MicroRNAs play central roles in animal development by binding 3’ untranslated regions of target mRNAs and inducing translational repression and/or degradation. Despite rapid advances in understanding microRNA biogenesis and function, less is known about how the activity of the microRNA effector complex, miRISC, is modulated. Specifically, post-translational modifications to miRISC and miRISC interactions with RNA binding proteins require further elucidation. This dissertation describes the potentiating role of the conserved serine/threonine kinase CK2 and the antagonizing function of the RNA binding protein CEY-1 on microRNA-mediated gene silencing in the nematode Caenorhabditis elegans.We identified casein kinase II (CK2) in a genome-wide screen to identify factors that regulate small RNA-mediated silencing pathways. Further genetic characterization revealed that CK2 promotes microRNA function in diverse cellular contexts. While CK2 is dispensable for microRNA biogenesis and stability of miRISC factors, it is required for efficient target binding and silencing. Importantly, we identified the conserved DEAD-box helicase, CGH-1/DDX6, as a CK2 substrate within miRISC and demonstrate that CK2-mediated phosphorylation of a conserved serine residue in CGH-1 is required for its function in the microRNA pathway. C. elegans Y-box protein-1 (CEY-1) is part of an ancient family of nucleic acid binding proteins that regulates mRNA stability and translation. We discovered loss of cey-1 potently suppressed lethal defects of a let-7 microRNA mutant. Furthermore, cey-1 mutation attenuates the increased expression of the let-7 target, lin-41 mRNA, but does not alter let-7 levels, indicating CEY-1 antagonizes let-7 miRISC target silencing but not let-7 biogenesis. Consistently, CEY-1 binding sites were identified in the 3’UTRs of let-7 targets, including lin-41, by high-throughput sequencing of RNA isolated by crosslinking and immunoprecipitation. Taken together, our data support a model where CEY-1 antagonizes let-7 pathway by direct binding of common mRNA substrates and positively regulating the stability and/or translation of let-7 targets. Dysregulation of microRNA activity is a hallmark of several common human diseases. Since CK2 and CEY-1 are both conserved in human, understanding their regulation of miRISC will better inform our understanding of human diseases and potentially aid in the development of microRNA-based therapeutics.

[发布机构] University of Michigan

[效力级别] RNA binding protein

[关键词] microRNA (miRNA);RNA binding protein;C. elegans genetics;CK2 (Casein kinase II);Genetics;Science;Human Genetics