The translationally active <i>Prm2</i> mRNA associates with the p42 CBF-A isoform.

<p>(<b>A–B</b>) Fractionation of adult mouse testicular lysates on a 15–50% continuous sucrose gradient in the absence (A) or presence (B) of EDTA. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA and on immunoblots for CBF-A (SAK22) and hnRNP A2. (<b>C–D</b>) The <i>Prm2</i> mRNA is translationally inhibited in postnatal day −28 and −30 mouse testis. (<b>C</b>) Developmental expression of <i>Prm2</i> mRNA in mouse testes. Total RNA was collected from mouse testis of 20, 28, 32 postnatal day of age (20–32 d), and analyzed by RT-PCR. RT, reverse transcriptase. (<b>D</b>) Developmental expression of PRM2 and CBF-A in mouse testes. Cytoplasmic testicular extracts from 20, 28, 30, 32 d mice were analyzed on immunoblots for PRM2, CBF-A (SAK22) and tubulin. (<b>E</b>) RIPs from adult and 30 d mouse testicular extracts. Cytoplasmic fractions were incubated with SAK22, ICCI or control anti-mouse IgGs. Immunoprecipitated fractions were analyzed on immunoblots for CBF-A (SAK22) and hnRNP A2, and by RT-PCR with primers amplifying the <i>Prm2</i>, α-tubulin or clusterin cDNAs. (<b>F–G</b>) The CBF-A p42 isoform associates with the 5′ mRNA cap complex. Cytoplasmic fraction from adult mouse testis was incubated with 7-methyl-GTP (m⁷GTP)-Sepharose or protein G-Sepharose (control). Bound proteins were analyzed on immunoblots for CBF-A (SAK22), hnRNP A2 and eIF4E and α-tubulin. (F) Lanes 1, 2 respectively 2% and 1% input. (G) Lane 1 and 2 are 2% input. Where indicated, testis lysates were pre-incubated with RNase A prior to incubation with the beads.</p

The <i>Prm2</i> mRNA expression is regulated by CBF-A at the translational level during spermatogenesis.

<p>(<b>A</b>) Immunoblots of CBF-A (Sak22), PRM2 and tubulin on Hnrnpab^+/− and Hnrnpab^−/− testis lysates. Total proteins were stained with Coomassie brilliant blue and shown as loading control. Densitometric analysis of the signals in the immunoblots was performed on 3 individual animals per genotypes. Data represent mean +/−SE ^*<i>p</i><0.05 student t-test. (<b>B</b>) Immunostaining for CBF-A on Hnrnpab^+/− and Hnrnpab^−/− testis sections. Bar, 50 µm. (<b>C</b>) Northern blotting analysis for the <i>Prm2</i> mRNA on total RNA from Hnrnpab^+/− and Hnrnpab^−/− mouse testis. Densitometric analysis was on 3 individual animals of each genotype, and shown as mean +/− SE. (<b>D</b>) <i>In situ</i> hybridization on Hnrnpab^−/− and Hnrnpab^+/− testis sections showed no differences on <i>Prm2</i> mRNA expression. Bar, 50 µm. The inset shows a magnified image of the marked area. (<b>E</b>) Fractionation of testicular lysates from adult Hnrnpab^+/+ and Hnrnpab^−/− mice on a 15–50% continuous sucrose gradient. Fractions were analyzed on Northern blots for the <i>Prm2</i> mRNA. (<b>F</b>) Testis section of Hnrnpab^+/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 was not detected in the seminiferous tubules of stage IX–XII, in which pachytene spermatocytes (P) and elongating spermatids (EL) are contained. (<b>G</b>) Testis section from Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). PRM2 signal was detected in some nuclei of elongating spermatids. (<b>H and I</b>) Magnified image of marked area in (G). DAPI staining shows that chromatin of PRM2-positive nuclei (white arrow head) appears more condensed than in PRM2-negative nuclei (blank arrow head). (<b>J</b>) Testis section of Hnrnpab^−/− stained with a PRM2 antibody (Green) and DAPI (gray). The seminiferous tubule was categorized as stage IX–XII, because it does not contain round-spermatid layer (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858.s007" target="_blank">Figure S7</a>), but nuclei of elongating spermatids appear prematurely condensed and positive for PRM2 signals. * denotes background staining of Leydig cells with the PRM2 antibody. Bars, 50 µm.</p

CBF-A is in cytoplasmic RNPs in complex with the <i>Prm2</i> mRNA in testicular cells.

<p>(<b>A</b>) Immunoblots of nuclear and cytoplasmic fractions of testis lysates. Anti-fibrillarin (nucleolar protein) and anti-Tom20 (mitochondrial protein) antibodies were used to characterize the fractionation. (<b>B</b>) Untreated or RNase-treated cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI) or control non-specific IgGs and the immunoprecipitates were probed as indicated. * marks a testis-specific variant of hnRNP A2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003858#pgen.1003858-Kamma1" target="_blank">[56]</a>. (<b>C</b>) RNA immunoprecipitation (RIP) assays. Cytoplasmic fractions of testis lysates were incubated with anti-CBF-A antibodies (SAK22 or ICCI), control anti-mouse IgGs, or without antibodies (mock). In all cases, total RNA was extracted from the immunoprecipitates and analyzed by RT-PCR with primers amplifying <i>Prm2</i>, α-tubulin or clusterin cDNA. (<b>D</b>) Densitometric quantifications of the RIP experiments. The signal intensities of the RT-PCR bands were calculated from 3 independent experiments and shown as % of input (mean+/−SE).</p

The RTS in the 3′ UTR of the <i>Prm2</i> mRNA is primarily targeted by p37.

<p>(<b>A</b>) Location and sequence of the <i>Prm2</i> mRNA RTS and control probes. For the pull-down assays all probes were biotinylated and individually coupled to streptavidin Sepharose. (<b>B–C</b>) <i>In vitro</i> RNA pull-down assays with recombinant (B) p37 and p42 or (C) p37 and hnRNP A2, incubated with wtRTS-Prm2 beads. As revealed on immunoblots with CBF-A (SAK22) or hnRNP A2 antibodies, all proteins were specifically co-precipitated with the beads. However, upon co-incubation with p37, precipitations of (B) p42 and (C) hnRNP A2 were significantly impaired. (<b>D</b>) p42 and hnRNP A2 are co-precipitated with wtRTS-Prm2 beads. (<b>E</b>) p37 specifically targets the RTS-containing <i>Prm2</i> mRNA 3′UTR. As indicated biotinylated oligonucleotides and <i>in vitro</i> transcribed full-length or Δ3′UTR <i>Prm2</i> mRNA were conjugated to Streptavidin beads and incubated with cytoplasmic testicular lysate. Bound proteins were analyzed by immunoblotting for CBF-A (SAK22 antibody) and hnRNP A2. Densitometric measurements of the bound CBF-A (<b>F</b>) and hnRNP A2 fractions (<b>G</b>) to biotinylated transcripts in panel (E) over three independent experiments are plotted as signal intensities from the respective immunoblots. The bar diagrams include standard deviations.</p