Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) Identification of YTHDC2-associated partners by immunoprecipitation (IP) experiments with either YTHDC2 or normal immunoglobulin antibodies from P12 mouse testes. Immunoprecipitated YTHDC2 protein complexes were separated by SDS-PAGE, and the gel was stained with silver before mass spectrometry (MS). Testis lysates were prepared from mouse at P12 ( n = 8). ( B ) Selective YTHDC2-associated protein candidates identified by MS analysis from P12 mouse testes. ( C ) Immunoprecipitation of YTHDC2 from mouse testis lysates at P21 and Western blot with anti-YTHDC2, anti-MEIOC, and anti-RBM46 antibodies (top). RBM46 protein was immunoprecipitated with anti-RBM46 antibodies with or without RNases, followed by Western blot analysis with indicated antibodies (bottom). ( D ) Full-length GFP- Rbm46 and FLAG- Ythdc2 or HA- Meioc expression constructs were cotransfected in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG or anti-HA antibodies and Western blot analysis with indicated antibodies. IB, immunoblotting. ( E ) GFP-tagged RBM46 mutants with deleted fragments were coexpressed with FLAG-YTHDC2 in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies before Western blot analysis with GFP antibodies. ( F ) GST pull-down assay showing that in vitro translated YTHDC2 does not bind to GST-RBM46. GST-RBM46 was expressed in E. coli and purified with glutathione beads. YTHDC2 with N-terminal His tag was in vitro translated. ( G and H ) Immunofluorescence analysis of Rbm46 -HA knock-in mice with anti-HA antibodies and the acrosome marker PNA that defines different stages of spermatogenesis. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. Lower panels show magnification image of single cell in the upper panels. Spg, spermatogonia; PL, pre-leptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene; RS, round spermatids; ES, elongating spermatids. Scale bars, 5 μm.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot, Expressing, Construct, Pull Down Assay, In Vitro, Purification, Immunofluorescence, Knock-In, Marker

Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) RBM46 and YTHDC2 exhibit a similar distribution pattern along the length of mRNA. ( B ) RBM46 eCLIP mRNA targets substantially overlapped with YTHDC2 mRNA targets by CLIP-seq in P10 testis. In total, 53 and 52% of P10 YTHDC2–binding sites were co-occupied by RBM46 within a 100- and 70-nt window, respectively. ( C ) Top motif at best-scored YTHDC-binding sites from P10 and adult YTHDC2 CLIP-seq using HOMER. Sequences within ±100 nt relative to YTHDC2-binding sites were used for de novo motif analysis. ( D ) RBM46-binding motif AAUCAUGU is the top motif within a 100-nt window centered on YTHDC2 U-rich motifs in both P10 and adult testis. Motif analysis by HOMER was performed with best-scored P10 and adult YTHDC2 CLIP targets. ( E ) Position and distance between RBM46 and YTHDC2 binding on their shared targets. ( F ) Schematic illustrations for the function of RBM46-MEIOC-YTHDC2 complex during spermatogenesis. RNA binding protein RBM46 forms an ancient posttranscriptional network with MEIOC and YTHDC2 to recognize and destabilize mitotic transcripts for a successful meiotic entry.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Binding Assay, RNA Binding Assay