Identification of a protein factor shared by the recycling pathways of U2- and U12-type spliceosomes
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Pre-mRNA splicing takes place in a large ribonucleoprotein complex, the spliceosome, which contains five small nuclear ribonucleoprotein complexes (snRNPs) and over 100 protein factors. There are two types of introns, the major- and the minor-type, that are excised by two different spliceosomes, assembled by two distinct sets of snRNPs: U1, U2, U4, and U6 snRNPs are required for splicing of the major-type introns; U11, U12, U4atac, and U6atac are their corresponding counterparts, involved in minor-type intron splicing. U5 snRNP is common for both spliceosomes. U4 and U6 snRNAs enter the spliceosome in the form of the U4/U6.U5 tri-snRNP, in which they are extensively base-paired. After splicing, U4 and U6 are released as singular snRNPs. In order to restore the tri-snRNP that can participate in another round of splicing, the U4/U6 hybrid must be recycled. The Prp24 protein in the yeast system, and its distant human homologue p110 have been identified as U4/U6 recycling factors and as specific protein components of U6 and U4/U6 snRNPs.This work focuses on the recycling the minor spliceosome and of the minor spliceosomal U4atac/U6atac snRNP. Using a recycling assay coupled to splicing in vitro, p110 was demonstrated to function also as U4atac/U6atac recycling factor. p110 was detected in U6atac snRNP. In contrast to the major U4/U6, however, it is associated only at background levels with U4atac/U6atac snRNP. Interestingly, U4/U6 and U4atac/U6atac hybrids can be also disrupted independently of splicing in vitro. This process may provide a mechanism to control splicing rates in vivo.The sequence elements of U6 and U6atac snRNA, required for interaction with p110 were identified: U6 nucleotides 38 to 57, and U6atac nucleotides 10 to 30 are sufficient for p110 binding. These regions are the most highly conserved between U6 and U6atac.Finally, singular U6 snRNP was purified from HeLa S100 extract. It contained a novel protein, p85 (cDNA GenBank accession AK001239), identified by mass-spectrometric analysis. p85 localizes to the nucleoplasm, and associates not only with U6, but also with U2 snRNA, and 5S and 5.8S rRNAs. This protein is weakly similar to the yeast rRNA processing factor NOP4, which implicates a function of p85 in maturation of U2 and U6 snRNAs.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Mol. Cell. Biol., 24 (2004), S. 1700-1708; EMBO J., 21 (2002), S. 2724-2735