The RNA helicase Prp43p is one of the few members of the DEAH-box helicase family that is known to operate in more than one cellular process in Saccharomyces cerevisiae. With roles in ribosome biogenesis and pre-mRNA splicing, Prp43p may be important in maintaining a communication conduit between these two pathways. Our studies provide insights into how Prp43p function is regulated through the use of three cofactors, Ntr1p, Pfa1p, and Gno1p, all of which interact with Prp43p at different steps of pre-mRNA splicing or ribosome biogenesis. Each cofactor contains a unique G-patch domain and our data show that they associate with Prp43p in a mutually exclusive manner. A strong growth defect and RNA processing phenotypes are seen upon overexpression of Pfa1p due to the dominance of Pfa1p interaction with Prp43p. Moreover, excess Pfa1p precludes Prp43p from interacting with either 35S pre-rRNA or U6 snRNA, indicating this one cofactor can negatively regulate Prp43p recruitment into ribosome biogenesis and pre-mRNA splicing pathways, respectively. We have determined that Ntr1p and Gno1p are able to compete with one another for Prp43p occupancy. Similar to Ntr1p, we show that the G-patch domain of Gno1p contributes to its association with Prp43p. To further understand pathway specificity of Prp43p, we characterized conditional prp43 alleles with mutations C-terminal to the conserved RecA domains of Prp43p. These novel alleles affect pre-mRNA splicing and ribosome biogenesis, though none are mutually exclusive. Multiple prp43 alleles are deficient in tri-snRNP formation, a previously uncharacterized phenotype in prp43 mutants. The majority of our prp43 mutants display varying rRNA defects, with some alleles impacting ribosome biogenesis more severely or moderately than known prp43 ATPase mutants. To correlate the processing defects seen in each allele, we have determined the extent of association of the mutants with each G-patch protein. Altogether, our data support a working model for Prp43p in which its substrate specificity, activation, and cellular distribution is coordinated through the efforts of the three G-patch proteins in yeast and sheds light on potential mechanisms of general DExH/D helicase function and regulation. / text

Pre-mRNA splicing by the spliceosome requires the precise and regulated efforts of the five snRNAs (U1, U2, U4, U5, and U6) and numerous associated proteins. Following assembly and activation of the spliceosome, two consecutive reactions result in intron removal and exon ligation from pre-mRNA substrates. It has been established that several members of the DExH/D-box family of helicases act transiently on the spliceosome prior to the chemical steps to authorize the successive reactions by hydrolyzing ATP and consequently inducing structural rearrangements. While it has been suggested that these changes produced in the structure of the spliceosome result in optimal positioning of the reactive species, the mechanisms and products of these reorganizations remain uncharacterized. The work presented here describes the genetic strategy for accumulating and purifying spliceosomes arrested in vivo, during the catalytic steps of the splicing cycle. Using these complexes, we have defined the components required to proceed through the first and second steps of splicing, in addition to the factors required for the release of the spliced message. Analysis of these functional, synchronized particles has also allowed us to define a function for Prp2p in initiating the first step of pre-mRNA splicing. Our data suggest that Prp2p may act in an ATP-independent manner to remodel the spliceosome prior to using its ATPase function to displace the SF3 complex. We propose that the SF3 complex, in addition to its role in identification of the branchpoint, also acts to sequester the reactive 2’OH of the branchpoint adenosine to prevent premature reactivity. Following the two catalytic steps of the splicing cycle, the spliceosome must disassemble and recycle its snRNPs for further rounds of splicing. The essential U6 snRNP component Prp24p, mediates one of the early assembly events - the annealing between the U4 and U6 snRNAs. We have discovered that although Prp24p is essential for viability, its function(s) can be bypassed by overexpressing the U6 snRNA. Additionally, biochemical characterizations of various forms of the U4/U6 snRNP provide evidence that Prp24p must be released before other components of the U4/U6 snRNP are permitted to interact and facilitate tri-snRNP formation. / text

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In eukaryotes, the spliceosome is a macromolecular ribonucleoprotein machine that excises introns from pre-mRNAs through two sequential transesterification reactions. The chemistry and fidelity of pre-mRNA splicing are dependent upon a series of spliceosomal rearrangements, which are mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily of proteins, whose members couple ATP hydrolysis to promote RNP structural rearrangements within the spliceosome. This is typified by Prp22, which promotes release of the spliced mRNA from the spliceosome and ensures fidelity of the second step of splicing. This role is well documented through classical biochemical and yeast genetics methods. Yet very little is known regarding the comings and goings of Prp22 relative to the spliceosome. My thesis research investigated the dynamics of Prp22 during splicing by using single-molecule fluorescence methods that allowed direct observation of these events. To do this, I helped construct a toolkit that combined yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS) with in vitro splicing assays. Specifically, my thesis research consisted of CoSMoS splicing experiments in which fluorescently labeled pre-mRNA, spliceosome components and Prp22 were directly visualized and analyzed. Using these methods, I found that Prp22’s interactions with the spliceosome are highly dynamic and reversible. By simultaneously monitoring Prp22 and individual spliceosome subcomplexes, I was able to frame these Prp22 binding events in context relative to specific steps in spliceosome assembly and splicing. These experiments provide insight into how Prp22 promotes mRNA release from the spliceosome and maintains splicing fidelity.

No description available.