Our laboratory is interested in the mechanisms of RNA-protein and RNA-RNA recognition. Currently, we are studying molecular interactions within the mammalian spliceosome, concentrating on interactions that define the pre-mRNA branch site and the structural dynamics of the spliceosome about this site. These studies should lead to significant insight into the exact roles of RNAs and proteins in the spliceosome, identify catalytic moieties, and extend our overall understanding of RNA-protein interactions and of ribonucleoprotein enzymes.

Background.ÝThe precise removal of intervening sequences is an essential step in the maturation of most eukaryotic pre-mRNAs and is an important control point for developmental and tissue-specific gene regulation. Such intron excision proceeds by way of two sequential transesterification reactions, or steps: 5' splice site cleavage/lariat formation and 3' splice site cleavage/ligation of the two exons. Both reactions take place within the spliceosome, a ribosome-sized complex composed of pre-mRNA, four small nuclear ribonucleoprotein particles, and many additional protein factors. Currently, most components are known, but what they all do and exactly how they function together are not at all clear.

RNA-protein interactions. Critical questions for understanding the splicing process are how the sites for chemical events are determined and how the reactions are catalyzed. Our research has involved analysis of the branch site, whose 2'-OH is the nucleophile that attacks the 5' splice site phosphate. Although the branch region is known to pair with U2 snRNA, no specific mechanism of nucleophile presentation is known. To examine its recognition and function, we undertook three lines of investigation: identifying closely juxtaposed proteins; determining the role of functional groups on the adenine base; and examination of the bulged disposition of the adenosine within the U2/branch site duplex. These studies detected new protein-RNA interactions and unanticipated spliceosomal rearrangements. One of these proteins, p14, interacts in a base-specific fashion and thus may mediate interactions with the branch site within spliceosomal complexes. Current objectives are to characterize these components (e.g., the p14 was previously unknown), interactions between them, and how these interactions impact on the exchange of one set of protein-RNA interactions for another as splicing proceeds. This will allow a detailed analysis of their function.  A new experimental approach will identify molecules specifically contacting the RNA substrate only after splicing catalysis has occurred, opening a new view into the core of the spliceosome.

Structural rearrangements and proof-reading. RNA helicases are likely to be involved in a variety of cellular processes involving RNPs. Multiple RNA-RNA rearrangements occur during spliceosomal assembly, most likely due to the activity of DEAD- and DEXH-type putative helicases. We have developed a novel system that reveals a dynamic exchange of U2 snRNP particles at inappropriate branch sites. This represents a biochemical approach to studying proof-reading steps by the spliceosome, and we are currently pursuing the factors responsible. The study of these factors will elucidate mechanisms of branch site proofreading, of RNA-RNA dynamics within the spliceosome, and of RNA helicases in general.Ý
 
 

Latest Publications
 

• Moore, M.J., Query, C.C. and Sharp, P.A. (1993). Splicing of precursors to mRNA by the spliceosome (Review). in: The RNA World, R. Gesteland and J. Atkins, eds. (New York: Cold Spring Harbor Laboratory Press), pp. 303-357.
• Query, C.C., Moore, M.J. and Sharp, P.A. (1994). Branch nucleophile selection in pre-mRNA splicing: Evidence for the bulged duplex model. Genes & Development 8, 587-597. [Abstract]
• MacMillan, A.M., Query, C.C., Allerson, C.R., Chen, S., Verdine, G.L. and Sharp, P.A. (1994). Dynamic association of proteins with the pre-mRNA branch region. Genes & Development 8, 3008-3020. [Abstract]
• Query, C.C., Strobel, S.A. and Sharp, P.A. (1996). Three recognition events at the branch site adenine. EMBO Journal 15, 1392-1402. [Abstract]
• Query, C.C., McCaw, P.S. and Sharp, P.A. (1997). A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract. Mol. Cell. Biology 17, 2944-2953.Ý [Abstract] [Full-Text Artcle]
• Moore, M.J. and Query, C.C. (1998). Use of site-specific modified RNAs constructed by ligation. In: RNA-Protein Interactions: A Practical Approach, C. Smith, ed. (Oxford: Oxford University Press), pp. 75-108.
• Moore, M.J. and Query, C.C. (2000). Joining of RNAs by splinted ligation. In: Methods in Enzymology, D.W. Celander and J.N. Abelson, eds. (Academic Press), 109-123.
• Will, C.L., Schneider, C., MacMillan, A.M., Katopodis, N.F., Neubauer, G., Wilm, M., Lührmann, 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. [Full-Text Artcle]
• 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. [Full-Text Artcle]
• Query, C.C. (2002). A glimpse of the catalytic core of a group II intron. Structure 10, 444-446.[Full-Text Artcle]
• 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. [Full-Text Artcle]
• Wang C, Query C.C., and Meier U.T. (2002). Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140. Mol Cell Biology 22, 8457-8466. [Full-Text Artcle]
  

Pre-1993 Publications

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