Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing.
In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing. Significantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally defined as U4/U6 snRNP-specific as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates. However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri-snRNP. Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease.
Makarova
Olga V
OV
Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Makarov
Evgeny M
EM
Liu
Sunbin
S
Vornlocher
Hans-Peter
HP
Lührmann
Reinhard
R
D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000235:genetics / Q000502:physiology*; D005656:Fungal Proteins; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D025941:Protein Interaction Mapping; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D012174:Retinitis Pigmentosa / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D017381:Spliceosomes / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D005136:Eye Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; C491451:PRP31 protein, S cerevisiae; C454222:PRPF31 protein, human; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins