Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading [sRNA-seq]

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Here, we report the identification of an evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an artificial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. The nuclear protein RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. RBV also promotes the loading of miRNAs into AGO1. In addition, RNA-seq revealed a global splicing defect in the mutant. Thus, this evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing. Overall design: small RNA profiles of 15-d-old Arabidopsis thaliana seedlings with amiR-SUL, amiR-SUL ab57, wild type (Columbia background)and ab57 in Col background input and after AGO1 IP were generated by deep sequencing, in triplicate, using Illumina HiSeq2500 and illumina Nextseq, respectively.

微小RNA（microRNAs，miRNAs）可通过RNA切割或翻译抑制的方式，在基因表达调控中发挥关键作用。本研究鉴定出一种进化保守的WD40结构域蛋白，其参与拟南芥中的miRNA生物发生过程。编码WD40结构域蛋白的褪绿叶脉区域减少（REDUCTION IN BLEACHED VEIN AREA，RBV）基因发生突变后，可抑制人工miRNA引发的叶片褪绿症状，同时使内源性miRNAs的积累呈现全局性下降。核蛋白RBV可通过提高RNA聚合酶II（RNA polymerase II，Pol II）在MIR基因启动子区域的结合占有率，促进MIR基因转录为初级miRNA（pri-miRNAs）。RBV还可促进miRNAs向AGO1的装载过程。此外，RNA测序（RNA-seq）结果显示，突变体中存在全局性的RNA剪接缺陷。综上，这种进化保守的核定位WD40结构域蛋白同时参与miRNA生物发生与RNA剪接过程。实验设计概要：本研究分别通过Illumina HiSeq2500与Illumina Nextseq测序平台，对三组生物学重复样本进行深度测序，以获取以下材料的小RNA（small RNA）图谱：携带amiR-SUL、amiR-SUL ab57的15日龄拟南芥幼苗，哥伦比亚生态型背景的野生型幼苗，以及哥伦比亚背景下ab57突变体的输入样本与AGO1免疫沉淀（AGO1 IP）产物。