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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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Sloan-Kettering Institute for Cancer Research
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