The primary transcripts of most eukaryotic genes (precursor mRNAs;pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing in a two-step pathway. Pre- mRNA splicing occurs in a ribonucleoprotein (RNP) complex called the spliceosome, which is composed of a large number of proteins and multiple U small nuclear RNP particles (U snRNPs). A small subset of introns, called U12-type introns (as opposed to the major class of U2-type introns), are spliced through the conventional two-step pathway but by a different spliceosome. The U2 snRNP Auxiliary Factor (U2AF) is an essential splicing factor, originally identified in our laboratory, that binds to the polypyrimidine (Py)-tract/3'splice site and initiates spliceosome assembly. Several human proteins are related to the small U2AF subunit, U2AF35, including a protein called U2AF35-related protein (Urp). During the past period of funding, we have shown that Urp is required for splicing of U12-type introns, and for the second step of U2- type intron splicing. In both cases, Urp directly contacts the 3'splice site. Using a combination of molecular biological, biochemical and structural approaches, we will continue to study how U2AF and Urp promote splicing. hUAP56, a member of the DExD/H-box family of RNA- dependent ATPases, was originally identified in our laboratory based upon its interaction with the large U2AF subunit (U2AF65). During the past period of funding we have shown that hUAP56 has multiple roles in U2-type splicing complex assembly, including disrupting the U2AF65-branchpoint/Py-tract interaction, interacting with U4 and U6 snRNAs, and unwinding of the U4/U6 snRNA duplex. We have also found that hUAP56 is required for splicing of U12-type introns. Experiments are proposed to understand the detailed mechanism by which hUAP56 facilitates diverse steps in splicing of U2- and U12-type introns. A variety of mammalian protein splicing factors contain an arginine-serine rich (RS) domain required to promote splicing. We have shown that direct contact with the branchpoint and 5'splice site is a general mechanism by which RS domains promote spliceosome assembly and splicing. Our studies have revealed a pathway of sequential interactions between RS domains and splicing signals during mammalian spliceosome assembly. Molecular, biochemical and structural experiments are proposed to understand in greater detail how RS domains are directed to splicing signals and promote spliceosome assembly and splicing, with an emphasis on studying the role of RS domain phosphorylation. Alternative splicing is an important mechanism of gene regulation and is responsible for substantially increasing diversity of the human proteome. However, the mechanisms that regulate alternative splicing remain largely unknown. During the past period of funding we have shown that FRG1, a protein whose over-expression is responsible for facioscapulohumeral muscular dystrophy (FSHD), functions by misregulating alternative splicing. Experiments are proposed to study how FRG1 affects splicing. In addition, we will perform genome-wide loss-of-function RNA interference screens to study mechanisms of splicing repression and to identify new splicing repressors.

Public Health Relevance

Pre-mRNA splicing is an essential step for expression of the vast majority of protein coding genes in higher eukaryotes. Disruption of normal pre-mRNA splicinggdue to mutations in either the cis-acting regulatory sequences required for splicing or the trans- acting components of the splicing machineryghas been shown to result in a variety of human diseases including atypical cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy and facioscapulohumeral muscular dystrophy, and has also been associated with cancer and metastasis. The experiments proposed in this application will increase our understanding of the factors and mechanisms involved in splice-site recognition, splicing complex assembly and splicing regulation, which may lead to the identification of new therapeutic targets and strategies for the treatment of diseases caused by aberrant pre-mRNA splicing. )

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Bender, Michael T
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University of Massachusetts Medical School Worcester
Schools of Medicine
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Misra, Ashish; Green, Michael R (2017) Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators. Methods Mol Biol 1507:1-12
Misra, Ashish; Green, Michael R (2016) From polyadenylation to splicing: Dual role for mRNA 3' end formation factors. RNA Biol 13:259-64
Park, Sung Mi; Ou, Jianhong; Chamberlain, Lynn et al. (2016) U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation. Mol Cell 62:479-90
Misra, Ashish; Ou, Jianhong; Zhu, Lihua J et al. (2015) Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors. Mol Cell 58:819-31
Misra, Ashish; Ou, Jianhong; Zhu, Lihua Julie et al. (2015) Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data. Genom Data 6:217-21
Moon, Heegyum; Cho, Sunghee; Loh, Tiing Jen et al. (2014) SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene. Biochim Biophys Acta 1839:1132-40
Jang, Ha Na; Lee, Minho; Loh, Tiing Jen et al. (2014) Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8. Biochim Biophys Acta 1839:25-32
Lin, Ling; Chamberlain, Lynn; Pak, Magnolia L et al. (2014) A large-scale RNAi-based mouse tumorigenesis screen identifies new lung cancer tumor suppressors that repress FGFR signaling. Cancer Discov 4:1168-81
Cho, Sunghee; Moon, Heegyum; Loh, Tiing Jen et al. (2014) PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion. Biochim Biophys Acta 1839:517-25
Loh, Tiing Jen; Moon, Heegyum; Cho, Sunghee et al. (2014) SC35 promotes splicing of the C5-V6-C6 isoform of CD44 pre-mRNA. Oncol Rep 31:273-9

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