Pre-mRNA splicing is an essential step in eukaryotic gene expression and serves as a key point of regulation. Splicing removes intervening intron sequences from precursors of messenger RNAs (pre- mRNA) to establish the correct reading frame for mRNA translation. Additionally, differential inclusion of exon coding sequences by alternative splicing vastly expands information potential of genes. Mutations that affect splicing are associated with a number of human diseases, including cancers. The goal of this proposal is to obtain and interpret structural information for the spliceosome, the very large macromolecular machine responsible for splicing catalysis. This information is necessary to understand how the spliceosome is able to precisely recognize very distant splice sites along a pre- mRNA and coordinate intron excision and exon ligation. Because the spliceosome is a dynamic complex composed of five structural RNAs (the U-rich small nuclear U1, U2, U4, U5 and U6 snRNAs) and over of 100 proteins, it presents special challenges to structural studies. We will combine sophisticated mass spectrometry approaches and electron microscopy studies to examine spliceosomes arrested at different stages relative to splicing chemistry. By comparing the different conformations, we will determine how the many spliceosome components are arranged and create models of spliceosome structure. These studies are critical to understanding mechanisms of splice site identification, spliceosome assembly, and splicing catalysis.

Public Health Relevance

The spliceosome is a critical molecular machine that precisely edits gene transcripts in a process is called splicing. Errors in splicing are associated with over 10% of human genetic disease, including cancers. Our goal is to model the three-dimensional structure of the spliceosome to understand how it functions. That understanding will be key to developing therapeutics for diseases that are caused by alterations in spliceosome function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Flicker, Paula F
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University of California Santa Cruz
Schools of Arts and Sciences
Santa Cruz
United States
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Effenberger, Kerstin A; Urabe, Veronica K; Jurica, Melissa S (2016) Modulating splicing with small molecular inhibitors of the spliceosome. Wiley Interdiscip Rev RNA :
Effenberger, Kerstin A; Urabe, Veronica K; Prichard, Beth E et al. (2016) Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages. RNA 22:350-9
Adams, B M; Coates, Miranda N; Jackson, S RaElle et al. (2015) Nuclear cyclophilins affect spliceosome assembly and function in vitro. Biochem J 469:223-33
Ilagan, Janine O; Jurica, Melissa S (2014) Isolation and accumulation of spliceosomal assembly intermediates. Methods Mol Biol 1126:179-92
Ilagan, Janine O; Chalkley, Robert J; Burlingame, A L et al. (2013) Rearrangements within human spliceosomes captured after exon ligation. RNA 19:400-12
Dudzik, Christopher G; Walter, Eric D; Abrams, Benjamin S et al. (2013) Coordination of copper to the membrane-bound form of ýý-synuclein. Biochemistry 52:53-60
Cvitkovic, Ivan; Jurica, Melissa S (2013) Spliceosome database: a tool for tracking components of the spliceosome. Nucleic Acids Res 41:D132-41
Coltri, Patricia; Effenberger, Kerstin; Chalkley, Robert J et al. (2011) Breaking up the C complex spliceosome shows stable association of proteins with the lariat intron intermediate. PLoS One 6:e19061
Roybal, Gabriel A; Jurica, Melissa S (2010) Spliceostatin A inhibits spliceosome assembly subsequent to prespliceosome formation. Nucleic Acids Res 38:6664-72
Ilagan, Janine; Yuh, Patrick; Chalkley, Robert J et al. (2009) The role of exon sequences in C complex spliceosome structure. J Mol Biol :

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