Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6middle dotU5 tri-snRNP formation and pre-mRNA splicing

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Abstract
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.