Journal: Genes

Article Title: Transcriptional Dysregulations of Seven Non-Differentially Expressed Genes as Biomarkers of Metastatic Colon Cancer

doi: 10.3390/genes14061138

Figure Lengend Snippet: Dark biomarkers of metastatic colon cancer were confirmed in three independent datasets. The “Dark biomarker” column lists the detected dark biomarkers as transcriptomic feature names. “Gene,” “Location,” and “Annotation” columns show the corresponding gene symbol, genomic location, and function annotations. The annotation data were collected from the GeneCards ( https://www.genecards.org/ , last accessed on 26 October 2022) [ 32 ] and UniProtKB ( https://www.uniprot.org/ , last accessed on 26 October 2022) [ 44 ] databases.

Article Snippet: Tanabe et al. observed that the protein level of YTHDC2 was positively correlated with CC tumor stage and metastasis [ ].

Techniques: Biomarker Discovery, Translocation Assay

Journal: Genes

Article Title: Transcriptional Dysregulations of Seven Non-Differentially Expressed Genes as Biomarkers of Metastatic Colon Cancer

doi: 10.3390/genes14061138

Figure Lengend Snippet: Summary of the lncRNAs overlapping with the seven detected dark biomarkers. The first two columns, “DB” and “Gene,” represent the dark biomarkers and their annotated genes. The dark biomarker 213212_x_at was annotated with four related genes. The next four columns, “Chr,” “Start,” “End,” and “S,” represent the specific position information of each gene on the human genome version 38. The last two columns provide the number of overlapping lncRNAs on the sense and antisense strands based on the LncBook database 2.0 [ 35 ].

Article Snippet: Tanabe et al. observed that the protein level of YTHDC2 was positively correlated with CC tumor stage and metastasis [ ].

Techniques: Biomarker Discovery

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