The primary transcripts of most eukaryotic structural genes (pre-mRNAs) contain intervening sequences that are removed by RNA splicing. The biochemical mechanisms involved in pre-mRNA splicing and in particular how these mechanisms allow accurate splice-site selection to be achieved are not fully understood. During the past several years we have developed an efficient and versatile system for studying pre-mRNA splicing in vitro. This approach has revealed new information about the biochemistry of pre-mRNA splicing and has allowed for the identification and characterization of probable splicing intermediates. Based upon these results, we have proposed a pathway for splicing of pre-mRNAs. In this proposal we outline experiments that continue and extend these studies. Our objectives are to determine the role of specific cis-acting pre-mRNA sequences in splicing, identify and characterize the biochemical factors that are involved in pre-mRNA splicing, and study the interaction of these factors with the RNA substrate. Finally, we will study in vitro the processing of adenovirus primary transcripts that undergo regulated differential splicing in vivo. By using extracts prepared from viral-infected cells, we hope to duplicate these alternative splicing events in vitro. Such a regulated in vitro processing system should provide a useful model for analyzing the biochemical mechanisms involved in the control of gene expression by differential RNA processing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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Harvard University
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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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