The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5' splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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