Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R.
T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.
Izquierdo
José M
JM
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, C/Nicolás Cabrera 1, Lab-107, Cantoblanco, 28049 Madrid, Spain. jmizquierdo@cbm.uam.es
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000954:Antigens, Surface / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005091:Exons; D057809:HEK293 Cells; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010455:Peptides / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D019014:fas Receptor / Q000235:genetics*
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D010455:Peptides; D016601:RNA-Binding Proteins; D019014:fas Receptor; C076751:TIAL1 protein, human