RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.This work was supported by grants from the National Health and Medical Research Council (NHMRC) to DJ (#606503); the Australian Research Council (ARC) to MKO and CJO; the New South Wales Cancer Council, Cancer Institute New South Wales, Banque Nationale de Paris-Paribas Australia and New Zealand, RT Hall Trust, and the National Breast Cancer Foundation to CJO. DJ was an NHMRC Peter Doherty Postdoctoral Fellow (#384297). MKO and CJO are NHMRC Senior Research Fellows (#545805, #481310). CCG is an NHMRC Australia Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Endometrial CRISP3 is regulated throughout the mouse estrous and human menstrual cycle and facilitates adhesion and proliferation of endometrial epithelial cells

The endometrium (the mucosal lining of the uterus) is a dynamic tissue that undergoes extensive remodeling, secretory transformation in preparation for implantation of an embryo, inflammatory and proteolytic activity during menstruation, and rapid postmenstrual repair. A plethora of local factors influence these processes. Recently, a cysteine-rich protein, CRISP3, a clade of the CRISP, antigen 5, pathogenesis-related (CAP) protein superfamily, has been implicated in uterine function. The localization, regulation, and potential function of CRISP3 in both the human and mouse endometrium is described. CRISP3 localizes to the luminal and glandular epithelium of the endometrium within both species, with increased immunoreactivity during the proliferative phase of the human cycle. CRISP3 also localizes to neutrophils, particularly within the premenstrual human endometrium and during the postbreakdown repair phase of a mouse model of endometrial breakdown and repair. Endometrial CRISP3 is produced by primary human endometrial epithelial cells and secreted in vivo to accumulate in the uterine cavity. Secreted CRISP3 is more abundant in uterine lavage fluid during the proliferative phase of the menstrual cycle. Human endometrial epithelial CRISP3 is present in both a glycosylated and a nonglycosylated form in vitro and in vivo. Treatment of endometrial epithelial cells in vitro with recombinant CRISP3 enhances both adhesion and proliferation. These data suggest roles for epithelial and neutrophil-derived CRISP3 in postmenstrual endometrial repair and regeneration.</p

RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing

RBM5 RRM2 is required for appropriate pre-mRNA splicing.

<p>(<b>A</b>) Relative abundance of <i>St5</i>, <i>Asb1</i> and <i>Plg2g10</i> is significantly increased in the RBM5 pulled down (RBM5-PD) samples compared to that of IgG-PD controls. Error bars = S.D. (standard deviation, <i>n</i> = 3 sets of each pull down). * indicates statistical significance (p&lt;0.05, t-test). (<b>B–D</b>) The R263P mutation in the RRM2 of RBM5 leads to aberrant splicing of <i>St5</i>, <i>Asb1</i> and <i>Pla2g10</i>. Black bars represent exons and open bars represent UTRs. FL: full-length, ex: exon, Δ ex 3 refers to exon 3 skipping, Δ ex 3+ex 4 refers to exon 3 and exon 4 skipping, and + intron refers to intron retention. (<b>E</b>) Splicing defects of <i>Kif17</i>, <i>Anks3</i>, <i>Rangap1</i>, <i>Nfx1</i> and <i>Cftr</i>. Transcripts with obvious shift in their relative intensities are indicated by arrows.</p

Functional clustering of differentially expressed genes identified by microarray analysis of RNAs isolated from round spermatids of <i>Rbm5^sda/sda</i> and <i>Rbm5^WT/WT</i> postnatal day 28 testes.

a<p>Differentially expressed genes associated with a particular significant function are given.</p

ST5 negatively regulates ERK signalling in round spermatids.

<p>(A) Schematic of the mouse ST5 protein. (B) Reduction of the ST5 protein leads to hyperactivation of ERK1/2 in round spermatids. HPRT was used a loading control. (C) ST5 localises strongly in the cytoplasm of round spermatids as determined by immunohistochemistry of postnatal day 28 testis section. (C, insert) No positive signal was observed when the ST5 antibody was omitted. Scale bars = 50 µM.</p

RBM5 target mRNAs in round spermatids identified using RNA pull down coupled with microarrays.

<p>RBM5 target mRNAs in round spermatids identified using RNA pull down coupled with microarrays.</p

<i>Rbm5</i> mRNA is highly expressed in the testis where the protein localises in somatic and germ cells.

<p>(<b>A</b>) <i>Rbm5</i> mRNA expression in various tissues and (<b>B</b>) during the establishment of the first wave of spermatogenesis. Sk: skeletal. Error bars = S.D. (standard deviation, <i>n</i> = 3 wild-type C57BL/6JxCBA mice, per age group) (<b>C</b>) RBM5 protein localisation in an adult <i>Rbm5^WT/WT</i> testis. (<b>C, inset</b>) RBM5 antibody pre-absorption control. Sg: Type A spermatogonia, PL: preleptotene spermatocyte, PS: pachytene spermatocyte, rST: round spermatid, Sp: spermatozoa, SC: Sertoli cell, PT: peritubular cell. Scale bars = 50 µm.</p

The <i>Rbm5^sda/sda</i> results in the substitution of an arginine for proline in the RBM5 RRM2.

<p>(<b>A</b>) Schematic of the mouse <i>Rbm5</i> gene. Black bars represent exons and open bars represent untranslated regions (UTRs). (<b>B</b>) Schematic of the mouse RBM5 protein. RS: arginine-serine domain; RRM: RNA recognition motif; ZF: zinc finger motif; OCRE: octamer repeat domain; G-patch: glycine patch domain, aa: amino acid (<b>C</b>) Sequence alignment of RRM2 from different species. (<b>D</b>) Cartoon representation of the RBM5 RRM2 structure as derived using NMR (PDBid: 2LKZ) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003628#pgen.1003628-Song1" target="_blank">[11]</a>. Amino acid residues within the ß2–ß4 strands that have been shown to be perturbed by RNA binding are shown in black including R263 in the ß2-strand.</p

Levels of serum testosterone, FSH and LH in adult <i>Rbm5^sda/sda</i> and <i>Rbm5^WT/WT</i> mice.

<p>Data are expressed as mean±S.D. (standard deviation). <i>n</i> = 5 per group, age 10–14 weeks old. p values&lt;0.05 were defined as being statistically significant (unpaired t-test, two-tailed).</p