Mutations in the MOV10L1 ATP Hydrolysis Motif Cause piRNA Biogenesis Failure and Male Sterility in Mice

Abstract

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs. piRNAs protect the genome integrity of the germline by silencing active transposable elements and are essential for germ cell development. Most piRNA pathway proteins are evolutionarily conserved. MOV10L1, a testis-specific RNA helicase, binds to piRNA precursors and is a master regulator of piRNA biogenesis in mouse. Here we report that mutation of the MOV10L1 ATP hydrolysis site leads to depletion of piRNAs on Piwi proteins, de-repression of transposable elements, and conglomeration of piRNA pathway proteins into polar granules. The Mov10l1 mutant mice exhibit meiotic arrest and male sterility. Our results show that mutation of the MOV10L1 ATP hydrolysis site perturbs piRNA biogenesis.

Keywords: MOV10L1; Male infertility; Meiosis; Retrotansposon; piRNA.

FIG. 1

Generation and expression of the Mov10l1 knockin allele with mutations in its ATP hydrolysis site. A) The RNA helicase domain of the MOV10L1 protein is shown. *ATP-binding (Walker A) and **ATP hydrolysis (Walker B) motifs. K(778) and D(888)E(889) are critical residues. The K778A knockin mutation was previously reported [43]. B) Generation of the Mov10l1 knockin (DE888.889AA) allele. The neo selection cassette is flanked by loxP sites and is removed by Actb-Cre. *Position of the DE → AA mutation in Mov10l1. C) Western blot analysis of MOV10L1 in testes from E16.5, Postnatal Day 6 (6d), 10d, 14d, and 18d and 8 wk-old Mov10l1^KI/+ and Mov10l1^KI/KI mice. ACTB serves as loading control. D) Semiquantitative RT-PCR analysis of Mov10l1 and Actb transcript abundance in testes from 10-day-old Mov10l1^KI/+ and Mov10l1^KI/KI mice. Total RNAs were pretreated with DNase I. RT-PCR produced no products in controls without reverse transcriptase (data not shown).

FIG. 2

Meiotic arrest in Mov10l1^KI/KI mice. A) Dramatic size reduction of testis from 6-wk-old Mov10l1^KI/KI mice. B and C) Histology of testes from 8-wk-old Mov10l1^KI/+ and Mov10l1^KI/KI mice. Two types of spermatocytes are present in the Mov10l1^KI/KI seminiferous tubules, indicated by arrowheads and arrows respectively. ES, elongating spermatids; RS, round spermatids; Spc, spermatocytes. Bars = 50 μm.

FIG. 3

MILI and MIWI2 are depleted of pre-pachytene piRNAs in embryonic Mov10l1^KI/KI testes. The MILI and MIWI2 complexes were immunoprecipitated from the testes of E16.5 Mov10l1^KI/+ and Mov10l1^KI/KI embryos. MILI-bound and MIWI2-bound RNAs (vertical lines) were isolated from MILI and MIWI2 complexes and radiolabeled for the detection of piRNAs. Lysate incubated with uncoupled beads served as a control. The RNAs associated with MILI in the Mov10l1^KI/KI testes were sequenced using HiSeq 2000 platform for 50 cycles.

FIG. 4

De-repression of IAP and LINE1 retrotransposons in Mov10l1^KI/KI gonocytes is shown. Sections of testes from E16.5 Mov10l1^KI/+ and Mov10l1^KI/KI embryos were immunostained with antibodies against (A and B) IAP GAG and (C and D) LINE1 ORF1p. DNA was stained with DAPI. Bar = 50 μm.

FIG. 5

De-repression of IAP and LINE1 retrotransposons in postnatal Mov10l1^KI/KI testes is shown. Sections of testes from 6-wk-old Mov10l1^KI/+ and Mov10l1^KI/KI mice were immunostained with antibodies against (A and B) IAP GAG and (C and D) LINE1 ORF1p. DNA was stained with DAPI. Spc, spermatocytes; Spg, spermatogonia. Bar = 50 μm.

FIG. 6

piRNA pathway proteins form polar conglomeration in embryonic Mov10l1^KI/KI gonocytes. Sections of testes from E16.5 Mov10l1^KI/+ and Mov10l1^KI/KI embryos were immunostained with antibodies against MIWI2 (A and B), MOV10L1 (C and D), and MILI (E and F). DNA was stained with DAPI. Bar = 50 μm.