The primary transcripts of most eukaryotic structural genes (pre- mRNAs) contain intervening sequences that are removed by RNA splicing. In some instances, alternative splicing of a common pre- mRNA provides an important mechanism for regulating gene expression. We have been studying the biochemical mechanisms involved in pre-mRNA splicing using in vitro systems. Splicing occurs in a large multicomponent complex, which contains both proteins and small nuclear ribonucleoprotein particles (snRNPs). It is likely that once this complex is formed, the splice sites that will ultimately be joined have already been determined. To study the early events involved in splicing complex assembly we will pursue two complementary approaches that have already been initiated in the laboratory. First, we will further purify the proteins involved in pre-mRNA splicing and characterize their mechanisms of action. In addition to proteins, the U1, U2, U5, and U4/U6 snRNPs are splicing factors. We will use methods recently developed in our laboratory for reconstituting all of these snRNPs from their isolated RNA moieties and protein components. This method should allow us to further study the interaction of snRNPs with the pre-mRNA and with each other during splicing complex assembly. In addition, we will initiate a dissection of each snRNP into functional domains by determining the snRNA regions and snRNP structural polypeptides required for the various snRNP activities. Finally, experiments are proposed to determine how splice sites are accurately selected and the mechanisms involved in regulated alternative splicing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035490-05
Application #
3288335
Study Section
Molecular Biology Study Section (MBY)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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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