Removal of intron regions from pre-messenger RNA (pre-mRNA) requires spliceosome assembly with pre-mRNA, then subsequent spliceosome remodeling to allow activation for the two steps of intron removal. Spliceosome remodeling is carried out through the action of DExD/H-box ATPases that modulate RNA-RNA and protein-RNA interactions. The ATPase Prp16 remodels the spliceosome between the first and second steps of splicing by catalyzing release of first step factors Yju2 and Cwc25 as well as destabilizing U2-U6 snRNA helix I. How Prp16 destabilizes U2-U6 helix I is not clear. We show that the NineTeen Complex (NTC) protein Cwc2 displays genetic interactions with the U6 ACAGAGA, the U6 internal stem loop (ISL) and the U2-U6 helix I, all RNA elements that form the spliceosome active site. We find that one function of Cwc2 is to stabilize U2-U6 snRNA helix I during splicing. Cwc2 also functionally cooperates with the NTC protein Isy1/NTC30. Mutation in Cwc2 can suppress the cold sensitive phenotype of the prp16-302 mutation indicating a functional link between Cwc2 and Prp16. Specifically the prp16-302 mutation in Prp16 stabilizes Cwc2 interactions with U6 snRNA and destabilizes Cwc2 interactions with pre-mRNA, indicating antagonistic functions of Cwc2 and Prp16. We propose that Cwc2 is a target for Prp16-mediated spliceosome remodeling during pre-mRNA splicing.
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| http://dx.doi.org/10.1093/nar/gku431 | DOI Listing |
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Article Synopsis
- The spliceosome's U2/U6 RNA core relies on both RNA interactions and spliceosomal proteins for its stable 3D structure.
- Mutations in certain U6 nucleotides showed that while some RNA interactions had minimal impact on splicing, others were crucial, especially U6-G60, whose removal completely halted splicing.
- The findings suggest that while RNA stacking interactions enhance coordination of metal M2 crucial for splicing, the overall function of the RNA core is supported by a combination of RNA-RNA and Protein-RNA interactions.
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