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, the pre-mRNA branch site, 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.
Orthogonal systems for in vivo investigation of catalytic center interactions. During pre-mRNA splicing, the branch site (BS) base pairs with a phylogenetically invariant sequence in U2 snRNA; this interaction is essential for both spliceosome assembly and first-step catalysis. Detailed investigation of the behavior/dynamics of the BS-U2 duplex has been limited by the highly deleterious nature of U2 mutations that disrupt BS-U2 pairing. Thus, we developed an orthogonal system wherein a dedicated second-copy U2 with a grossly substituted BS-binding sequence mediates the splicing of a cognate reporter gene. This orthogonal BS-U2 pair produces a non-essential second spliceosome that allows in vivo characterization of the BS-U2 helix, positioning of the first-step nucleophile, and its interaction with the spliceosome core, with few constraints. These properties allowed us to demonstrate that the BS-U2 structure exists at the time of first-step catalysis.
Currently, little is known of the second-step catalytic core. We are using our orthogonal systems to elucidate the 3'SS binding site within the second-step core. These experiments demonstrate that the branch structure formed in the first step translocates on the triplet repeat sequence in U2 (GUAGUA) and the 3'SS then binds in a geometry analogous to that of the first-step nucleophile. The introduction of additional complementary sets of mutations will allow further mechanistic investigation of the second-step core.