Identification of a 35S U4/U6.U5 Tri-snRNP Complex Intermediate in Spliceosome Assembly [RNA]

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 of zinc finger and a G-patch domain as we characterized previously, regulates pre-mRNA splicing in a RNA binding-independent manner. We found that ZIP is physically associated with the U4/U6.U5 tri-snRNP. Remarkably, ZIP-containing tri-snRNP has a sedimentation coefficient ~35S, a tri-snRNP that has not been described before. We showed that the 35S tri-snRNP contains hPrp24, indicative of a state when the U4/U6 di-snRNP is just 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, whose 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.

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