Pre-mRNA processing is an essential step in eukaryotic gene expression. Constitutive splicing of intervening sequences (introns) from precursors of messenger RNAs (pre-mRNAs) is necessary to establish the correct reading frame for translation. Additionally, alternative inclusion of different coding sequences (exons) from the same transcript places splicing as a pivotal point of gene regulation. Mutations affecting both constitutive and alternative 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. A three-dimensional (3D) structural understanding of this important molecule will be necessary to elucidate how this dynamic complex 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 on the order of 100 proteins, it presents challenges to structural studies. Cryo-electron microscopy (cryo-EM) provides a means to visualize this complicated machine. We will pursue a combination of EM labeling and biochemical characterization of purified splicesomes arrested between the two chemical steps of splicing chemistry to provide an interpretation of the cryo-EM structure. This will allow us to map spliceosome components on the structure to identify the pre-mRNA substrate and active site. These studies will move us closer to defining the mechanisms of splice site identification, spliceosome assembly, and splicing catalysis.
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| Effenberger, Kerstin A; Urabe, Veronica K; Jurica, Melissa S (2017) Modulating splicing with small molecular inhibitors of the spliceosome. Wiley Interdiscip Rev RNA 8: |
| 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 |
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| 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 |
| Cvitkovic, Ivan; Jurica, Melissa S (2013) Spliceosome database: a tool for tracking components of the spliceosome. Nucleic Acids Res 41:D132-41 |
| 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 |
| 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 |
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