Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. As many as one third of all heritable disease mutations result in splicing defects. To better understand the mechanism of splicing we propose a large-scale implementation of classic in vitro splicing assays and assembly assays. We propose to use this technology to map splicing elements around the 3'ss, to map branchpoints upstream of the 3'ss and also to repurpose spent exome sequencing libraries as in-vitro splicing substrates. This final goal stages an in-vitro splicing assay on characterized, genotyped samples with the aim of extracting phenotypic data from a genotyping tool. The existence of millions of characterized sequencing libraries could potentially connect in-vitro discovery to clinically relevant patient diagnosis.

Public Health Relevance

A great deal of research effort has been invested in understanding how genes are expressed. In vivo profiling experiments have recorded which transcripts accumulate in cells across different tissues. As part of the technological revolution of the post genomic era, these measurements are made at a grand scale (i.e. thousands of transcripts followed simultaneously). However the mechanisms of splicing have been determined for only a few model substrates. Here, we remedy this deficiency by implementing in-vitro mechanistic studies at a genomic scale. We propose to map functional elements and rare branched intermediates for all the introns in the genome. These in vitro recordings will provide a vital piece of information to complement in vivo studies and help us better understand biology and disease.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HG007905-01
Application #
8765808
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Pazin, Michael J
Project Start
2014-07-14
Project End
2016-06-30
Budget Start
2014-07-14
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Providence
State
RI
Country
United States
Zip Code
02912
Soemedi, Rachel; Cygan, Kamil J; Rhine, Christy L et al. (2017) The effects of structure on pre-mRNA processing and stability. Methods 125:36-44
Cygan, Kamil J; Soemedi, Rachel; Rhine, Christy L et al. (2017) Defective splicing of the RB1 transcript is the dominant cause of retinoblastomas. Hum Genet 136:1303-1312
Taggart, Allison J; Lin, Chien-Ling; Shrestha, Barsha et al. (2017) Large-scale analysis of branchpoint usage across species and cell lines. Genome Res 27:639-649
Soemedi, Rachel; Cygan, Kamil J; Rhine, Christy L et al. (2017) Pathogenic variants that alter protein code often disrupt splicing. Nat Genet 49:848-855
Lin, Chien-Ling; Taggart, Allison J; Lim, Kian Huat et al. (2016) RNA structure replaces the need for U2AF2 in splicing. Genome Res 26:12-23
Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G (2016) RNA structure in splicing: An evolutionary perspective. RNA Biol 13:766-71
Fredericks, Alger M; Cygan, Kamil J; Brown, Brian A et al. (2015) RNA-Binding Proteins: Splicing Factors and Disease. Biomolecules 5:893-909