線蟲體內DEAD-box解旋酶和微小核糖核酸let-7基因間交互作用分析

Abstract

RNA helicases of the DEAD-box protein family have been shown to participate in many pathways that are associated with RNA structural conformation changes and RNA-protein interaction but their roles in microRNA processing and function remain unclear. Here we show an RNAi screen in the model organism C. elegans, taking the advantages of C. elegans genetics analysis and RNA interference tools, for DEAD-box RNA helicases that are involved in microRNA processing and/or function. First we searched for the proteins containing conserved sequences of DEAD-box helicases in C. elegans and then executed RNA interference to knock down their coding genes for reverse genetics analysis. We focused on the heterochronic phenotypes of the let-7 microRNA, such as vulva bursting, seam cell developmental defects and retarded adult gene expression, since the phenotypes are clear and have been used in several successful screens of microRNA regulators or targets in previous studies. We have screened 48 of 65 DEAD-box RNA helicases in C. elegans and found seven DEAD-box helicases: F01F1.7, cgh-1, vbh-1, F58E10.3, mog-4, H27M09.1, Y65B4A.6, whose RNAi knock-down caused let-7 loss-of-function phenotypes, may play a role in let-7 function. Analysis of genetic interactions of these DEAD-box RNA helicases and several alleles directing weak to moderate let-7 mutant phenotypes, including a weak allele let-7(mg279), a temperature-sensitive allele let-7(n2853), a null Argonaute mutant alg-1(gk214) and a deletion of two let-7 family miRNAs mir-48; mir-241(nDf51), have shown synergistic severities of heterochronic phenotypes in different levels. Moreover, reduced expression of F01F1.7, F58E10.3, mog-4 and Y65B4A.6 resulted in a decrease of the let-7 miRNA, suggesting a role of these DEAD-box RNA helicases in microRNA biogenesis. We have raised a rabbit polyclonal antibody against F58E10.3 and successfully expressed a HA-tagged F58E10.3 in C. elegans for future studies on the role of F58E10.3 in let-7 biogenesis and/or function by biochemical approaches.