Journal: Oncogene
Article Title: Emerging roles of RNA-binding protein hnRNPM in alternative splicing regulation and UTMD mediated sh-hnRNPM/CMBs suppress the proliferation of colorectal cancer
doi: 10.1038/s41388-025-03599-3
Figure Lengend Snippet: A , B The indicated siRNAs were transiently transfected into RKO cells, and the RNAs were extracted for RT-PCR analysis of PLEKHB2 exon 8 inclusion/exclusion ( A ). Quantification of each PSI value from RT-PCR results was presented as mean ± SD in the bar graph ( B ). ns no significance, *** p < 0.001 . C Upper: The schematic diagram of PLEKHB2 minigene. The alternative exon 8 is marked in orange. Bottom: RT-PCR analysis using RNA from RKO cells co-transfected with the indicated siRNAs, overexpression plasmids and PLEKHB2 minigene (PLEKHB2 minigene + ). PSI values were shown at the bottom of the gel. D Schematic diagram of RRM domains in hnRNPM, and constructions of three hnRNPM mutants: ΔRRM1 (deleting RRM1 domain), ΔRRM2 (deleting RRM2 domain), ΔRRM3 (deleting RRM3 domain). E 293 T cells were transiently overexpressed hnRNPM-HA, the RRM1, RRM2, or RRM3 deletion mutant plasmids. Western blot of the exogenous hnRNPM with anti-HA antibody and endogenous hnRNPM with anti-hnRNPM antibody. All the mutants and hnRNPM-WT plasmids were HA tagged. F Diagram for the specific primers designed in accordance with exon-intron boundary sequences to detect exon 7, 8, and 9. G , H RT-PCR analysis ( G ) and qRT-PCR assay ( H ) following CLIP assay indicated the direct binding between RRM domains and endogenous PLEKHB2 RNA fragments. PLEKHB2 pleckstrin homology domain containing B2, hnRNPM heterogeneous nuclear ribonucleoprotein M, RT-PCR reverse transcription PCR, qRT-PCR quantitative reverse transcriptase PCR, PSI percent of splicing in, RRM RNA recognition motif, CLIP crosslinking immunoprecipitation.
Article Snippet: Co-immunoprecipitation (Co-IP) assay was performed using Immunoprecipitation (Co-IP) Kit (MCE), Protein A/G Magnetic Beads (MCE) and Anti-HA tag antibody (Abcam, ab9110).
Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Mutagenesis, Western Blot, Quantitative RT-PCR, Binding Assay, Reverse Transcription, Cross-linking Immunoprecipitation
