The 5'm7GppN cap is a distinctive feature of eukaryotic mRNA that is required for mRNA stability and translation. The mRNA cap is formed by three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7) methyltransferase. Our long-term goals are to understand the mechanisms and specificities of the capping enzymes, illuminate how capping is coupled to transcription through interactions of the capping enzymes with components of the Pol II elongation complex, and explore the capping enzymes as anti-infective drug targets. Our studies have revealed differences in the structures, mechanisms, and inhibition profiles of capping enzymes from different taxa that recommend RNA triphosphatase and cap (guanine-N7) methyltransferase as targets for antifungal/antiprotozoal drug discovery - a major """"""""unmet need"""""""" in public health and infection control worldwide. Here we propose to uncover the basis for the exquisite sensitivity of the fungal cap guanine-N7 methyltransferase to the natural product sinefungin and to develop a bisubstrate transition-state analog built on a sinefungin scaffold. We've shown that the capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II, which is composed of a tandem array of YSPTSPS heptapeptide repeats. Capping enzymes also interact physically with elongation factor Spt5 and the CTD kinase Cdk9. This interaction network suggests an """"""""elongation checkpoint"""""""" that recruits the capping enzymes in a timely fashion and thereby avoid wasteful rounds of transcription of uncapped pre-mRNAs. We propose to dissect these interactions genetically and biochemically in order to test key predictions of the checkpoint model. A related goal is to explore the how the effector functions of the Pol II CTD are modulated by CTD-specific phosphatases. We focus on the essential phosphatases SpFcpl and Ssu72, which display a preference for Ser2-P and Ser5-P, respectively. A partial deficiency of human Fcp1 is associated with a genetic developmental disorder. We are poised to determine the atomic structure of ScFcpl. We have initiated a new line of research into the formation of the 2,2,7-trimethylguanosine (TMG) cap structure found on many small nuclear RNAs. We characterized Tgs1 and Tgs2 as cap-specific guanine-N2 methyltransferases.
We aim to elucidate their mechanism and structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052470-16
Application #
7809672
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
1995-05-01
Project End
2012-02-09
Budget Start
2010-05-01
Budget End
2012-02-09
Support Year
16
Fiscal Year
2010
Total Cost
$494,998
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Schwer, Beate; Sanchez, Ana M; Garg, Angad et al. (2017) Defining the DNA Binding Site Recognized by the Fission Yeast Zn2Cys6 Transcription Factor Pho7 and Its Role in Phosphate Homeostasis. MBio 8:
Schwer, Beate; Roth, Allen J; Shuman, Stewart (2017) Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2. RNA 23:420-430
Chatterjee, Debashree; Sanchez, Ana M; Goldgur, Yehuda et al. (2016) Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast. RNA 22:1011-25
Inada, Maki; Nichols, Robert J; Parsa, Jahan-Yar et al. (2016) Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast. Nucleic Acids Res 44:9180-9189
Agarwal, Radhika; Schwer, Beate; Shuman, Stewart (2016) Structure-function analysis and genetic interactions of the Luc7 subunit of the Saccharomyces cerevisiae U1 snRNP. RNA 22:1302-10
Smith, Paul; Ho, C Kiong; Takagi, Yuko et al. (2016) Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase. MBio 7:e00058-16
Schwer, Beate; Kruchten, Joshua; Shuman, Stewart (2016) Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring. RNA 22:1320-8
Mbogning, Jean; Pagé, Viviane; Burston, Jillian et al. (2015) Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification. Nucleic Acids Res 43:9766-75
Fotopoulos, Pauline; Kim, Jeongho; Hyun, Moonjung et al. (2015) DPY-17 and MUA-3 Interact for Connective Tissue-Like Tissue Integrity in Caenorhabditis elegans: A Model for Marfan Syndrome. G3 (Bethesda) 5:1371-8
Schwer, Beate; Ghosh, Agnidipta; Sanchez, Ana M et al. (2015) Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1. RNA 21:1135-46

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