The work in my laboratory focuses on two major steps in gene expression, mRNA splicing and mRNA export. Our long-term goal is to understand at a molecular level the mechanisms responsible for specificity and fidelity in these pathways. Our future work will address three fundamental questions: 1) How are the activities of the spliceosomal NTPases specifically regulated? A long-standing question is how the spliceosomal DEAD-box ATPases are activated at precise times in the splicing cycle. We recently identified a region of the U5 snRNP protein Prp8 that specifically stimulates the Brr2 ATPase to unwind U4 from U6 snRNA, the key event in catalytic activation of the spliceosome. Using activity- and FRET-based assays, we will now identify the full set of molecular interactions that control this step. We will focus on the roles of the positive activator Snu114, an EF2-like GTPase, and the proposed down-regulation of Brr2 by ubiquitylation of a Prp8-interacting factor. 2) How is transcription coupled to mRNA splicing and export? While it is apparent that the nuclear steps in gene expression are temporally coupled, little is understood about the underlying mechanistic bases of this coupling. We are employing an innovative high-throughput genetic platform to facilitate identification of quantitative genetic interactions;such Epistasis Mini-Array Profiles have proven powerful predictors of novel functional relationships. We will test specific predictions from our ongoing analysis that suggest unexpected connections between the proteasome and the nuclear pore, and between the spliceosome and the chromatin remodeling machinery. 3) How is splicing regulated in response to the environment? Using a global microarray-based assay, we recently demonstrated that amino acid starvation selectively inhibits the splicing of ribosomal protein gene transcripts. We will now determine the molecular basis of the novel signal transduction pathway mediating this response. We will also expand our battery of stressors to identify other splicing regulatory modules. More broadly, we will interrogate the biological impact of yeast introns by the quantitative analysis of each of ~270 strains engineered to contain a precise intron deletion.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021119-37
Application #
7796541
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1977-02-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
37
Fiscal Year
2010
Total Cost
$828,875
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Nissen, Kelly E; Homer, Christina M; Ryan, Colm J et al. (2017) The histone variant H2A.Z promotes splicing of weak introns. Genes Dev 31:688-701
Mayerle, Megan; Guthrie, Christine (2017) Genetics and biochemistry remain essential in the structural era of the spliceosome. Methods 125:3-9
Mayerle, Megan; Raghavan, Madhura; Ledoux, Sarah et al. (2017) Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency. Proc Natl Acad Sci U S A 114:4739-4744
Mayerle, Megan; Guthrie, Christine (2016) A new communication hub in the RNA world. Nat Struct Mol Biol 23:189-90
Mayerle, Megan; Guthrie, Christine (2016) Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing. RNA 22:793-809
Ledoux, Sarah; Guthrie, Christine (2016) Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity. J Biol Chem 291:11954-65
Soucek, Sharon; Zeng, Yi; Bellur, Deepti L et al. (2016) The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA. Mol Cell Biol :
Patrick, Kristin L; Ryan, Colm J; Xu, Jiewei et al. (2015) Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast. PLoS Genet 11:e1005074
Lipp, Jesse J; Marvin, Michael C; Shokat, Kevan M et al. (2015) SR protein kinases promote splicing of nonconsensus introns. Nat Struct Mol Biol 22:611-7
Holmes, Rebecca K; Tuck, Alex C; Zhu, Chenchen et al. (2015) Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough. PLoS Genet 11:e1005735

Showing the most recent 10 out of 101 publications