Levels of <i>Etv5</i> mRNA in postnatal day 3 testes (A), kidney (B) and spleen (C) in <i>Etv5^sco/sco</i> and wild-type (WT) mice.

<p>(D) ETV5 immunoblotting showed an absence of full length and truncated proteins in the mutant spleen. HPRT was used as a loading control. Levels of <i>Cxcr4</i> (E) and <i>Ccl9</i> (F) mRNAs in <i>Etv5^sco/sco</i> and WT postnatal day 3 testes. A–C, E–F, WT values were set as 100%. <i>n</i> = 3 mice per group, **p&lt;0.01, ***p&lt;0.001 (unpaired <i>t</i>-test, two-tailed).</p

<i>Etv5</i> mutant (<i>Etv5^sco/sco</i>) males are sterile due to the progressive loss of germ cells.

<p>(A) Testis of 8 weeks-old <i>Etv5^sco/sco</i> and a wild-type (WT) littermate. (B) Testis weight to body weight ratio of <i>Etv5^sco/sco</i> mice compared to WT littermates. <i>n</i> = 12 per group. *p&lt;0.05 (unpaired <i>t</i>-test, two-tailed). (C–F) PAS staining of 4–8 weeks-old testes of <i>Etv5^sco/sco</i> and WT mice.</p

The SCO mouse line carries a missense mutation within the <i>Etv5</i> gene.

<p>(A) Schematic of the mouse <i>Etv5</i> gene and the location of the identified mutation. Exon positions are based on the ENSMUST00000079601 transcript. (B) ETV5 protein and the location of the missense amino acid.</p

Phenotypic defects observed in homozygous mice from the <i>Etv5</i> allelic series.

<p>n/a: not analysed due to embryonic lethality.</p

Incidence of developmental abnormalities in <i>Etv5^sco/sco</i> adult mice.

<p>Incidence of developmental abnormalities in <i>Etv5^sco/sco</i> adult mice.</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

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

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

<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