The m7GpppN cap is a defining feature of eukaryal messenger RNA that is required for mRNA stability and efficient translation. Our long-term goal has been to understand the mechanism of cap formation and how capping is coupled to transcription. Capping entails three enzymatic reactions: (i) the 5'triphosphate end of the pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase (TPase);(ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase (GTase);and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (MTase). The capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II), which is composed of a tandem array of heptapeptide repeats (consensus: Y1S2P3T4S5P6S7). The inherently plastic CTD structure is sculpted by dynamic phosphorylation and dephosphorylation of the heptad serine residues. The CTD structure transmits informational cues about the state of the transcription machinery (a CTD code) that is "read" by CTD receptors. Our goal is to understand how CTD information content is specified and conveyed to CTD receptor proteins (especially the capping enzymes). Our genetic dissection of what is essential for fission yeast CTD function is providing new insights to the CTD code, including: (i) structure-activity relations at essential residues;(ii) the distinctive roles of position- specific phosphorylations in sexual differentiation and vegetative growth;and (iii) the ability to bypass "CTD pathologies" caused by S2A and S5A mutations. We've thereby demonstrated that the essential function of the Ser5-P mark in vivo is to recruit the capping enzymes. Capping enzymes also bind to the essential Pol II elongation factor Spt5, which, in conjunction with Spt4, elicits an elongation arrest at promoter-proximal sites that provides a temporal window for capping of nascent mRNAs. Fission yeast Spt5 binds to the capping enzymes via a distinctive "Spt5 CTD" composed of 18 repeats of a nonapeptide motif (consensus: T1P2A3W4N5S6G7S8K9). Genetic studies indicate that the CTDs of fission yeast Pol II and Spt5 play overlapping roles in recruiting the capping enzymes in vivo. This project aims to dissect the functions and structure-activity relations of the Pol II and Spt5 CTDs and the impact of CTD mutations on gene expression. We will determine structures of capping enzymes in complexes with the Pol II and Spt5 CTDs, and then assess genetically the functions of the capping enzyme-CTD interfaces. Capping enzymes discriminate different Pol II CTD phosphorylation arrays, implying that remodeling of the CTD by protein kinases and phosphatases is a means to regulate mRNA processing. Our biochemical and crystallographic studies of S. pombe Fcp1, an essential CTD phosphatase that preferentially hydrolyzes Ser2- PO4, are yielding deep insights to catalysis of CTD dephosphorylation in vitro. In this project, we propose to dissect genetically and biochemically the phosphoprotein substrate specificity of S. pombe Fcp1 and the requirements for Fcp1 function in vivo.

Public Health Relevance

mRNA capping enzymes are attractive targets for anti-infective drug discovery in light of the stark differences in the structures, mechanisms, and pharmacological sensitivities of the capping apparatus in humans versus fungal and protozoan pathogens. CTD phosphorylation dynamics orchestrate eukaryal gene expression. Two of the enzymes we study that modulate CTD structure are implicated in human pathology. A partial deficiency of human CTD phosphatase Fcp1 is associated with the autosomal recessive developmental disorder characterized by cataracts, facial dysmorphism, and peripheral neuropathy. Cdk9 CTD kinase activity is critical for HIV replication and Cdk9 dysregulation is associated with cardiac hypertrophy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052470-18
Application #
8423682
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
1995-05-01
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
18
Fiscal Year
2013
Total Cost
$521,422
Indirect Cost
$156,775
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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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
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
Schwer, Beate; Sanchez, Ana M; Shuman, Stewart (2015) RNA polymerase II CTD phospho-sites Ser5 and Ser7 govern phosphate homeostasis in fission yeast. RNA 21:1770-80

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