A Mammalian Germ Cell-Specific RNA-Binding Protein Interacts with Ubiquitously Expressed Proteins Involved in Splice Site Selection

• Published in: Proceedings of the National Academy of Sciences - PNAS 2000-05, Vol.97 (11), p.5717-5722
• Main Authors: Elliott, David J., Bourgeois, Cyril F., Klink, Albrecht, Stevenin, James, Cooke, Howard J.
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Biochemistry; Biological Sciences; Cell Nucleus - metabolism; Genes; Germ cells; Germ Cells - metabolism; Humans; Infertility, Male - metabolism; Male; Mice; Molecular interactions; Nuclear Proteins - metabolism; Peptide Fragments - genetics; Peptide Fragments - metabolism; Phosphoproteins - metabolism; Protein Binding; Protein Structure, Tertiary; Proteins; RBM gene; RBM protein; Recombinant Fusion Proteins - metabolism; Ribonucleic acid; RNA; RNA Precursors - metabolism; RNA Splicing; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Serine-Arginine Splicing Factors; Social interaction; Splicing; splicing factors; Substrate Specificity; Thioredoxin; Treaties; Two-Hybrid System Techniques
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.11.5717

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.