Fig. 4.
MOV10L1 interacts with Piwi proteins and is necessary for piRNA-dependent repression of retrotransposons. (A and B) Up-regulation of retrotransposons in Mov10l1−/− mice. (A) Microarray analysis of pooled total RNA (n = 3) from testes of Mov10l1−/− mice versus Mov10l1+/− mice at postnatal days 10, 12, and 14. The depicted retrotransposon families from the LINE and LTR class were among the strongest up-regulated transcripts in Mov10l1−/− mice at P14. (B) Immunofluorescent staining of testis sections from P14 mice for the LINE ORF1 protein (ORF1p, green); counterstaining of nuclei with DAPI (blue). (C) Total RNA of testis from 24-d-old Mov10l1+/− mice versus Mov10l1−/− mice was end-labeled with [32P]ATP and detected after denaturing PAGE. In Mov10l1−/− mice, ∼29-nucleotide-long RNAs, most likely representing piRNAs, were strongly reduced. (D) Total RNA from testis of P14 and P24 Mov10l1+/− versus Mov10l1−/− mice was isolated and used for Northern blotting to detect specific piRNAs (piR-4868 and piR-1) and miRNAs (let-7a). An ethidum bromide-stained polyacrylamide gel is shown as a loading control. (E) Detection of the Piwi proteins Mili and Miwi (white) in testis sections from P14 Mov10l1+/− versus Mov10l1−/− mice by immunofluorescent staining. (F) COS1 cells were cotransfected with expression plasmids for Flag-tagged MOV10L1 and Myc-tagged MILI and MIWI, respectively. Lysates were then used for coimmunoprecipitation with anti-Flag antibodies followed by Western blot analysis with anti-Myc antibodies. Input lanes represent 1% of lysates used for coimmunoprecipitation. The experiment was repeated three times; representative immunoblots are shown. Reciprocal coimmunoprecipitation worked similarly.