Charles C. Query, M.D., Ph.D.
Our laboratory is interested in mechanisms by which the spliceosome assembles and functions. We have focused on interactions that assemble splicing complexes around one of the chemical substrates ¨C the pre-mRNA branch site ¨C and on subsequent structural dynamics about this site. These studies are providing insights into the "mechanics" of a large RNP machine, the roles of helicases, and RNA-protein interactions.
Background. Precise intron removal is an essential maturation step for eukaryotic pre-mRNAs and a control point for regulation. Intron excision proceeds by two reactions catalyzed by the spliceosome, a 50-60S complex composed of five snRNAs and >100 proteins. How do these parts of the spliceosome "machine" work together? How is splicing modulated?
How is the pre-mRNA Engaged by snRNPs?
Structural Rearrangements in Pre-Spliceosome Assembly. At least eight RNA-dependent ATPase/helicases are required during spliceosome assembly and function, although how each ATPase/helicase effects rearrangements and alters interaction of snRNPs with the pre-mRNA is not yet understood. We are studying the first ATP-dependent step in spliceosome assembly and the required ATPase, Prp5, as a model rearrangement event. We find that not only does Prp5 hydrolyze ATP to mediate the interaction of U2 snRNP with the intron branch site, but it also is a bridge that provides cross-intron interaction between U1 and U2 snRNPs during pre-spliceosome formation. We are interested in what exactly does Prp5 move or position during this process and are purifying complexes both prior to and after ATP hydrolysis by Prp5 for biochemical comparisons and cryo-EM imaging.
How Can Catalytic Activity Be Modulated?
Many splice/branch sites (MOST in mammalian cells!) are not the optimal sequences. How can they be used for splicing? We carried out an open screen in S. cerevisiae for suppressors of a severe intron mutation and identified several spliceosomal proteins that strongly improve the ability of the spliceosome to use substrates containing mutations. Our analysis of these and previously identified suppressors led to a new and unified model by which all known suppressors act ¨C that suppression of substrate mutations results from altering the equilibrium between spliceosome conformations. This resembles tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity. Similar modulation of substrate selectivity may explain the ability of mammalian spliceosomes to act on the typically poor splice sites of alternatively spliced introns.
- Yang, F., Wang, X.-Y., Zang, Z.-M., Pu, J., Fan, Y.-J., Zhou, J., Query, C.C., and Xu, Y.-Z. (2013). Splicing proofreading at 5' splice sites by ATPaase Prp28p. Nucleic Acids Research doi:10.1093/nar/gkt149.
- Query, C.C. and Konarska, M.M. (2013). Spliceosome's core exposed. Nature 493, 615-616. doi:10.1038/nature11857.
- Shao, W., Kim, H.-S., Cao, Y., Xu, Y.-Z. and Query, C.C. (2012). A U1-U2 snRNP interaction network during intron definition. Mol Cell Biology 32, 470-478.
- Query, C.C. and Konarska, M.M. (2012). CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome. RNA 18, 1001-1013.
- Trcek, T., Larson, D.R., Moldon, A., Query, C.C., and Singer, R.H. (2011). Single molecule mRNA decay measurements reveal promoter regulated mRNA stability in yeast. Cell 147, 1484-1497.
- Moldon, A., and Query, C. (2010). Crossing the Exon. Molecular Cell 38, 159-161.
- Smith, D.J., Konarska, M.M., and Query, C.C. (2009). Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Molecular Cell 34, 333-343.
- Query, C.C. (2009). Spliceosome subunit revealed. Nature 458, 418-419.Â
- Smith, D., Query, C.C., and Konarska, M.M. (2008). 'Nought may endure but mutability': Spliceosome Dynamics and the Regulation of Splicing. Molecular Cell 30, 657-666.
- Xu, Y.-Z. and Query, C.C. (2007). Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly. Molecular Cell 28, 838-849.
- Smith, D.S., Query, C.C., and Konarska, M.M. (2007). Â trans-Splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing. Molecular Cell 26, 883-890.
- Liu, L., Query, C.C., and Konarska, M.M. (2007). Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nature Structural & Molecular Biology 14, 519-526.
- Query, C.C. and Konarska, M.M. (2006). Splicing fidelity revisited. Nature Structural & Molecular Biology 13, 472-474.
- Konarska, M.M., Vilardell, J., and Query, C.C. (2006). Repositioning of the reaction intermediate within the catalytic center of the spliceosome. Molecular Cell 21, 543-553.
- Konarska, M.M. and Query, C.C. (2005). Insights into the mechanisms of splicing: more lessons from the ribosome. Genes & Dev. 19, 2255-2260.
- Query, C.C. and Konarska, M.M. (2004). Suppression of a broad spectrum of substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Molecular Cell 14, 343-354.
- Xu, Y.-Z., Newnham, C.M., Kameoka, S., Huang, T., Konarska, M.M., and Query, C.C. (2004). Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA. EMBO Journal 23, 376-385.
- Query, C.C. (2002). A glimpse of the catalytic core of a group II intron. Structure 10, 444-446.
- Wang, C., Query, C.C., and Meier, U.T. (2002). Immunopurified snoRNPs pseudouridylate ribosomal RNA independently of their association with phosphorylated Nopp140. Mol. Cell. Biology 22, 8457-8466.
- Huang, T., Vilardell, J., and Query, C.C. (2002). Pre-spliceosome formation in S. pombe requires a stable complex of SF1-U2AF59-U2AF23. EMBO Journal 21, 5516-5526.
- Newnham, C.M., and Query, C.C. (2001). The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site. RNA 7, 1298-1309.
- Will, C.L., Schneider, C. MacMillan, A.M., Katopodis, N.F., Neubauer, G., Wilm, M., Luhrmann, R., and Query, C.C. (2001). A novel U2 and U11/12 protein that associates with the pre-mRNA branch site. EMBO Journal 20, 4536-4546.
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