The majority of human genes contain at least one intron that must be precisely spliced to avoid introducing errors during gene expression. Disruption of normal splicing patterns can cause or modify human disease. Spliceosome activation is the final regulatory step before catalysis, and therefore must be strictly controlledto ensure splicing fidelity. Addition of the U4/U6-U5 tri-snRNP to the spliceosome during spliceosome activation instigates a large number of RNA:RNA rearrangements, especially in snRNA U6. U5 helicase Brr2 unwinds the U4/U6 snRNA duplex allowing catalytically necessary structures like the U6 internal stem-loop (ISL) to form. The Prp8 Jab1/MPN domain modulates the RNA helicase activities of Brr2. Disruption of spliceosome activation can cause disease, as occurs in Retinitis pigmentosa (RP). RP (incidence 1/3500) is characterized by progressive retinal degeneration that ultimately proceeds to total blindness. Core splicing machinery components of the U4/U6-U5 tri-snRNP, particularly mutations in PRPC8 and SNRNP200, the human orthologs of yeast tri-snRNP proteins Prp8 and Brr2, have been linked to RP however how these mutant alleles affect splicing to cause disease is unknown. In particular, while it is known that Brr2 unwinds U4/U6 during spliceosome activation and that the Prp8 Jab1/MPN domain affects Brr2 activity, the kinetics and specific conformational rearrangements undergone by U6 during activation remain unknown, as are the molecular effects of RP alleles on these processes. Previous investigations were hindered by the fact that most splicing studies use crude splicing extract, wherein the many reversible steps in spliceosome assembly proceed asynchronously. To address this issue single-molecule FRET (smFRET) approaches to monitor splicing have been developed. smFRET provides a method to monitor dynam- ics and conformational rearrangements without the need to isolate or synchronize intermediates. The experi- ments described within this proposal first establish a smFRET assay to monitor U4/U6 unwinding in purified tri- snRNPs (Aim 1) and then use this system to determine if Brr2 and Prp8 RP mutants are misregulated in the assembled spliceosome (Aim 2). Further experiments will determine how splicing efficiency and fidelity is af- fected by RP mutants. Together this work will provide essential fundamental information on spliceosome activa- tion and how aberrant activation causes disease.

Public Health Relevance

The removal of introns from pre-mRNA by the spliceosome is one of the essential steps of gene expression in eukaryotes. Errors in intron removal are the cause of most hereditary human diseases, and are involved in the progression of metabolic disorders and cancers. The goal of the proposed research is to determine how con- formational changes in the spliceosome help ensure that splicing occurs without errors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Reddy, Michael K
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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