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. 2019 May 14;17(1):39.
doi: 10.1186/s12915-019-0659-z.

Biological and RNA Regulatory Function of MOV10 in Mammalian Germ Cells

Affiliations
Free PMC article

Biological and RNA Regulatory Function of MOV10 in Mammalian Germ Cells

Kaiqiang Fu et al. BMC Biol. .
Free PMC article

Abstract

Background: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells.

Results: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites.

Conclusions: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.

Keywords: MOV10; MOV10L1; Male germ cells; RNA helicase; RNA-binding protein; Spermatogonia; Splicing; Testis; miRNA; piRNA.

Conflict of interest statement

Ethics approval

All experiments involving mice were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Expression and localization patterns of MOV10 in male germ cells. a Time-course western blot analysis of MOV10 in postnatal mouse testes, compared with MOV10L1 and LIN28. TUBULIN was used as a loading control. Mouse testes were collected from P2 through adulthood. b Expression patterns of MOV10, MOV10L1, and LIN28 in different populations of male germ cells. Spg, spermatogonia; PS, pachytene spermatocytes; RS, round spermatids. c Western blot analysis of MOV10 in cytoplasmic and nuclear fractions of P10 testis. GAPDH and H3 were used as cytoplasmic and nuclear markers, respectively. MILI and MVH are known to be present in perinuclear granules in the cytoplasm. d Whole-mount immunostaining for MOV10 and PLZF in seminiferous tubules. Image shows As, Apr, and Aal (A4, A8, A16) clusters of spermatogonia that are interconnected by intercellular cytoplasmic bridges due to incomplete cytokinesis. Dotted lines mark clusters of PLZF (shown in red) and MOV10 (shown in green) double-positive cells, counterstained with DAPI (shown in blue). Scale bars, 50 μm. eg Frozen sections of testes from P10 (e), Spg isolated from P6–8 testes (f), and in vitro cultured SPCs (g) were co-immunostained for MOV10 and PLZF. Panel e shows high-magnification images of regions marked by dashed lines. Scale bar, 20 μm
Fig. 2
Fig. 2
Mov10 knockdown affects cell fate decisions in SPC. a Morphological differences between Mov10 shRNA (shMov10-832 or shMov10-833) and control (shVector) virus transduced SPC cultures at 6 days following lentiviral transduction. Mov10 shRNA cultures exhibit dissociation of clump-forming cells and contain only few and small grape-shaped colonies. Scale bar, 20 μm. b Significantly lower cell numbers in SPC cultures transduced with Mov10 shRNA vs cells transduced with control virus. c, d Flow cytometric analysis of apoptosis (c) and cell cycle (d). The error bars in b, c, and d represent variation (SEM) among biological triplicates. e, f Reduced repopulation capacity of SPCs after Mov10 knockdown. Mov10 shRNA or control vector transduced cells were transplanted into total 13 recipient testes. Donor-derived spermatogenic colonies (beta-glucuronidase transgene positive) were visualized by X-gal staining and counted. Scale bar, 2 mm. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3
Fig. 3
Mov10 knockdown affects the production of miRNA. a Read length profile for small RNAs showing that the percentage of small RNA reads were downregulated after Mov10 shRNA treatment. b Scatter plot of miRNA expression data from small RNA-seq represented as log2 values of Mov10 knockdown versus control samples. c qPCR validation of selected miRNAs with well-known function in SPCs. d Schematic representation of a general step-wise miRNA processing from pri- to mature forms (left) and the corresponding read pick-up from RNA-seq library and small RNA library (right). See the rationale in the Methods. e Scatter plot showing change of the transcript of each downregulated miRNA represented as Mov10 knockdown versus control samples. Results are mean values from three biological replicate RNA-seq libraries. f Distribution of downregulated miRNAs according to genomic origin of their mature form. g Analysis of intron-derived miRNAs using the same method as in panel E. h UCSC visualization of the genomic window of ± 200 nt flanking the hairpin of miR21a and miR20a shows lower RNA-seq read density in Mov10 knockdown SPC versus vector control (left) and qPCR confirmed significant reduction of miR21a and miR20a primary transcripts (right). PCR primer sets are marked by arrows relative to the position to the pre-miRNA shown in green. i The long isoform Vmp1-201 (ENSMUST00000018315.9) bearing the long 3′-UTR contains pre-miR21a (green line) and can be processed to produce the short isoform Vmp1-202 (ENSMUST00000123590.7) and miR21a precursor for downstream miRNA processing (left). Exons (black box), the alternative part of 3′-UTR (white box) as well as the common part (gray box) of 3′-UTR are shown. Two different primer sets (arrows) were designed. The relative ratio of the long isoform over both (long+short) was quantified by Image J software (right)
Fig. 4
Fig. 4
MOV10 regulates mirtron splicing in SPCs. PCR test of mirtron splicing events in SPCs and NIH/3 T3 cells. Total RNA samples from Mov10 knockdown versus control SPCs were examined by semi-quantitative RT-PCR analyses. The diagrams depict two splicing isoforms, black boxes represent exons, gray lines represent introns, green lines represent miRNAs, and arrows mark the positions of forward (F) and reverse (R) primers. E, exon number. The percent of intron retention (PIR) value, used as an indication for the extent of alteration of a splicing event, was determined as the proportion of the intron retention isoform versus the total level of intron retention and intron exclusion isoforms. The change of miRNA transcripts was calculated as the relative whole level of intron retention and intron exclusion isoforms in Mov10 knockdown over control. PCR products were quantified by Image J software. Results represent data from biological triplicates
Fig. 5
Fig. 5
Genomic mapping of in vivo captured MOV10 CLIP targetome. a Schematic of the HITS-CLIP procedure. HITS-CLIP employs the following steps to achieve target specificity: covalent crosslinking via ultraviolet irradiation (254 nm), disruption of protein-protein association by highly stringent wash, gel separation of protein-RNA ribonucleoproteins (RNPs) followed by membrane transfer, RNA retrieval, and expansion for deep sequencing. b Western blot and autoradiography of MOV10-RNA CLIP complexes. Non-crosslinked testes and IgG CLIP served as negative controls. Three independent libraries were prepared from RNA extracted from gel purified RNPs (marked with red line). c Percentage of CLIP reads mapping to genic and intergenic regions. d Percentage of CLIP reads mapping to genomic repeat sequences. The error bars in panel c and d represent variation among three independent CLIP libraries. e, f Genome browser view of CLIP reads over the genomic window for pre-pachytene piRNA cluster 3 (e) and cluster 5 (f). The main peak on the left of the piRNA cluster 5 likely reflects 3′-UTR of upstream mRNAs
Fig. 6
Fig. 6
Transcriptome-wide annotation and analyses of MOV10 target RNAs. a Distribution of MOV10 CLIP reads mapping to different regions of mRNAs. Three curves represent three CLIP libraries. b Distribution of MOV10 CLIP tags mapping to mRNAs with different 3′-UTR length. mRNAs were ranked by 3′-UTR length and divided into terciles containing equal transcript numbers. c The pie chart shows the distribution of RNA types identified as MOV10 CLIP targets. d Classification of CLIP reads into 5 categories (I–V) based on their relative position to pre-miRNA hairpins. Read distribution of categories I, II+III, and IV+V is calculated. e Normalized coverage of MOV10 CLIP reads (red curve) and secondary structure potential of corresponding genomic sequences (blue curve) around pre-miRNAs. MOV10 CLIP tags mapping to ± 200 nt windows flanking the middle point of pre-miRNAs are plotted as density values at single-nucleotide resolution. f Numbers of mature miRNA, pre-miRNA, and pri-miRNA bound by MOV10. Overlapping regions represent the miRNAs that are defined as no less than two forms. g Genome browser view and validation of MOV10-bound nuclear lncRNAs. UCSC visualization of MOV10 CLIP reads on the lncRNAs Malat1 and Neat1. The green bars represent three isoforms of Neat1. RT-PCR confirms nuclear localization of these lncRNAs in P10 testis. The amplicons of pre-GAPDH and GAPDH-intron serve as controls for nuclear fraction and GAPDH-exon serves as control for cytoplasmic fraction. The interaction between MOV10 and lncRNAs was validated by RIP-PCR. h MOV10-crosslinked positions within introns. The distribution of genomic crosslinked sites within intronic regions upstream and downstream of the splice site is shown. A genomic crosslinked site was defined as the corresponding genomic position of a deletion identified in a CLIP tag. Each unique position is only represented once
Fig. 7
Fig. 7
Identification of MOV10-interacting proteins in the nucleus. Western blot (a) and silver staining (b) of the MOV10 IP complex from nuclear lysate. c Gene ontology analysis of MOV10-associated proteins in the nucleus. d Nine MOV10-associated splicing-related proteins were selected for validation by IP-western blot assay. The piRNA pathway proteins MILI and MOV10L1 served as negative controls. e Venn diagram showing the cross analysis of four sets of MOV10 nuclear IP-MS data. The purple line marks 32 proteins that were reproducibly identified in IP using RIPA buffer without RNase treatment and at least one other IP with RNase treatment. f Gene ontology analysis of the 32 reproducible MOV10-assoicated proteins. g FLAG-MOV10 and HA-target (SRSF1, DDX5 or DDX17) were overexpressed in HEK293T cells, and IPs were performed by FLAG and HA antibodies, followed by western blot analysis
Fig. 8
Fig. 8
Subcellular function of MOV10 in germ cells. Diagram of proposed RNA regulatory mechanisms executed by MOV10 in interaction with diverse RNA species and multiple proteins. MOV10-mediated RNA regulation pathways are initiated in the nucleus, where MOV10 associates with splicing factors, such as SRSF1 and DDX5, to bind intronic regions of pre-mRNA. The splicing process is mechanistically coupled with transcription and RNA processing, linking directly or indirectly to the fate of nascent transcripts for miRNA, lncRNA, mRNA, or even 3′-UTR. In the case of Mov10 deficiency, this coordinated regulation is severely impaired, globally impairing RNA splicing/processing activity, and thus failure to maintain a normal balance of various RNA pools, for example, as illustrated here, MOV10-mediated splicing regulation of intronic miRNAs. MOV10 transports its target RNAs from the nucleus to the cytoplasm to carry out further maturation, degradation, or translation. MOV10 predominantly binds long 3′-UTR as a mediator of mRNA decay via interaction with the NMD factor UPF1. Thus, the disruption of MOV10-mediated RNA homeostasis could reflect a profound, combinatorial effect resulting from a cascade of hierarchical events causing RNA dysregulation

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