Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

Paula Stein,Gregory J Hannon,Nikolay V Rozhkov,Olga Davydenk,Richard M Schultz,Lee E Vandivier,Fan Li,Fabián L Cárdenas,Brian D Gregory,Paula E Cohen

doi:10.1371/journal.pgen.1005013

Abstract

The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

Highlights

The RNase III enzyme DICER is responsible for biosynthesis of short-interfering RNAs and microRNAs
This study unequivocally demonstrates an essential function for siRNAs in mammals, mediated through endonucleolytic cleavage of targets, and provides an explanation for the selective pressure that one AGO protein retains catalytic activity
DICER processes long double-stranded RNA precursors into 21–23 bp-long duplexes known as siRNAs [1]. miRNAs are encoded by specific genomic loci and are processed from endogenous hairpin-shaped transcripts that are initially cleaved in the nucleus to a 70-bp miRNA precursor by the Microprocessor complex, which is composed of the RNase III enzyme DROSHA and its partner, DiGeorge syndrome critical region 8 (DGCR8)

Summary

Introduction

The RNase III enzyme DICER is responsible for biosynthesis of short-interfering RNAs (siRNAs) and microRNAs (miRNAs). By utilizing an allele of Ago2 that can bind small RNAs, but is catalytically inactive, we show that spindle formation and chromosome congression depend on the action of endo-siRNAs. Live imaging technology revealed that the defects start during GVBD, when chromosomes and microtubule organizing centers (MTOCs) scatter instead of forming a sphere [16], resulting in a long, abnormal spindle with unaligned chromosomes that fail to progress to anaphase I.

Results

Conclusion