Abstract
The de novo assembly and post-splicing reassembly of the U4/U6.U5 tri-snRNP remain to be investigated. We report here that ZIP, a protein containing a CCCH-type zinc finger and a G-patch domain, as characterized by us previously, regulates pre-mRNA splicing independent of RNA binding. We found that ZIP physically associates with the U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP). Remarkably, the ZIP-containing tri-snRNP, which has a sedimentation coefficient of ∼35S, is a tri-snRNP that has not been described previously. We also found that the 35S tri-snRNP contains hPrp24, indicative of a state in which the U4/U6 di-snRNP is integrating with the U5 snRNP. We found that the 35S tri-snRNP is enriched in the Cajal body, indicating that it is an assembly intermediate during 25S tri-snRNP maturation. We showed that the 35S tri-snRNP also contains hPrp43, in which ATPase/RNA helicase activities are stimulated by ZIP. Our study identified, for the first time, a tri-snRNP intermediate, shedding new light on the de novo assembly and recycling of the U4/U6.U5 tri-snRNP.
Keywords:
Cajal body; RNA helicase; RNA splicing; alternative splicing; ribonuclear protein (RNP); snRNP assembly; spliceosome; tri-snRNP.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
MeSH terms
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Alternative Splicing*
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Antigens, Neoplasm / chemistry
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Antigens, Neoplasm / genetics
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Antigens, Neoplasm / metabolism*
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Coiled Bodies / chemistry
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Coiled Bodies / enzymology
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Coiled Bodies / metabolism
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HeLa Cells
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Humans
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Immunoprecipitation
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MCF-7 Cells
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Microscopy, Electron, Transmission
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Microscopy, Fluorescence
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Molecular Weight
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Mutation
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Negative Staining
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Oligopeptides / genetics
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Oligopeptides / metabolism
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Organelle Biogenesis*
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Protein Multimerization
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Protein Stability
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RNA Helicases / chemistry
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RNA Helicases / genetics
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RNA Helicases / metabolism*
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism*
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Ribonucleoprotein, U5 Small Nuclear / chemistry
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Ribonucleoprotein, U5 Small Nuclear / metabolism
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Spliceosomes / chemistry
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Spliceosomes / enzymology
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Spliceosomes / metabolism*
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Ubiquitin-Specific Proteases / chemistry
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Ubiquitin-Specific Proteases / genetics
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Ubiquitin-Specific Proteases / metabolism*
Substances
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Antigens, Neoplasm
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Oligopeptides
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Repressor Proteins
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Ribonucleoprotein, U5 Small Nuclear
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SART3 protein, human
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ZGPAT protein, human
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FLAG peptide
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DHX15 protein, human
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USP39 protein, human
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Ubiquitin-Specific Proteases
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RNA Helicases