The function and stability of eukaryotic RNA is regulated by co- and post-transcriptional modifications that include mRNA capping, splicing, and polyadenylation as well as processing and polyadenylation of non-coding RNA. The 5'm7GpppN cap is the first co-transcriptional mRNA modification and is required for pre-mRNA splicing, export, stability, and translation. Capping activities are recruited to the transcription apparatus through interactions with RNA polymerase II (RNAP II) after it is phosphorylated on serine residues within its C-terminal tandem heptapeptide repeats with the consensus sequence Y1S2P3T4S5P6S7. Capping enzymes also interact with the transcription elongation factor Spt5 and this occurs in fission yeast within the Spt5 C-terminal tandem nonapeptide repeats with the consensus sequence T1P2A3W4N5S6G7S8K9. The observation that capping enzymes are recruited to Spt5 prior to elongation at promoter proximal sites suggests that the transcription elongation checkpoint may entail coordinated recruitment of the capping apparatus.
In Aim 1 we will determine the x-ray structure of a mammalian capping enzyme RNAP II CTD complex and extend the resolution of structures of the yeast capping apparatus in complex with intact RNAP II.
In Aim 3 we will probe structure activity relationships for the fission yeast capping enzymes in complex with both RNAP II CTD and Spt5 CTD to reveal how capping enzymes interact with the transcription apparatus prior to and during early elongation. Results in Aims 1 and 3 will guide mutational analysis to probe the importance of interactions observed in our structures through biochemical and genetic analysis in budding and fission yeast. The transcription cycle is regulated by kinases and phosphatases that generate waves of phosphorylation and dephosphorylation that control the temporal and spatial patterning of RNAP II and Spt5 CTD phosphorylation. The Fcp1 RNAP II CTD phosphatase is essential and conserved across evolution. Fcp1 can dephosphorylate the RNAP II CTD at S2 and S5 positions although it is unclear if both activities are essential in vivo. We recently discovered S. pombe Fcp1 isoforms with altered CTD phosphatase specificities and in Aim 2 we will extend these studies through structural and genetic analysis in S. pombe to determine the effects of altered Fcp1 specificity on its essential functions. Non-coding RNA is also subject to co- and post-transcriptional modifications and the recently identified TRAMP complex promotes processing or decay through polyadenylation and interaction with the RNA exosome, a 3'-5'exoribonuclease.
In Aim 4, we will characterize TRAMP components by expressing, purifying, and reconstituting complexes that contain the Mtr4 RNA helicase, the Trf poly(A) polymerases and the Air RNA binding proteins. These preparations will be used in conjunction with our reconstituted RNA exosomes in biochemical assays to determine substrate specificities and activities for TRAMP in RNA processing and decay. Crystallization trials will be conducted with the long-term goal of illuminating the structural basis for TRAMP mediated polyadenylation.

Public Health Relevance

The function and stability of eukaryotic RNA is regulated in part by co- and post-transcriptional modifications that include capping, splicing, and polyadenylation. Co-transcriptional RNA processing begins the moment nascent RNA emerges from RNA polymerase and post-transcriptional RNA processing continues throughout the lifetime of a particular RNA. This proposal seeks to explore the role of RNA processing and decay factors that contribute to co-transcriptional 5'capping, RNA polymerase II CTD metabolism (a key regulator of co- transcriptional RNA processing), and nuclear RNA surveillance through characterization of the TRAMP complex. Pathways and enzymes that regulate the abundance, lifetime, and processing of RNA have been associated with human diseases that include neurodegenerative disorders, cancer, and inflammation.

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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Flicker, Paula F
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Sloan-Kettering Institute for Cancer Research
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Doamekpor, Selom K; Lee, Joong-Won; Hepowit, Nathaniel L et al. (2016) Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits. Proc Natl Acad Sci U S A 113:E4151-60
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
Doamekpor, Selom K; Schwer, Beate; Sanchez, Ana M et al. (2015) Fission yeast RNA triphosphatase reads an Spt5 CTD code. RNA 21:113-23
Doamekpor, Selom K; Sanchez, Ana M; Schwer, Beate et al. (2014) How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. Genes Dev 28:1323-36
Lyumkis, Dmitry; Doamekpor, Selom K; Bengtson, Mario H et al. (2013) Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase. Proc Natl Acad Sci U S A 110:1702-7
Ghosh, Agnidipta; Shuman, Stewart; Lima, Christopher D (2011) Structural insights to how mammalian capping enzyme reads the CTD code. Mol Cell 43:299-310
Ghosh, Agnidipta; Lima, Christopher D (2010) Enzymology of RNA cap synthesis. Wiley Interdiscip Rev RNA 1:152-72
Gu, Meigang; Rajashankar, Kanagalaghatta R; Lima, Christopher D (2010) Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA capping apparatus. Structure 18:216-27
Suh, Man-Hee; Meyer, Peter A; Gu, Meigang et al. (2010) A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme. J Biol Chem 285:34027-38
Ghosh, Agnidipta; Shuman, Stewart; Lima, Christopher D (2008) The structure of Fcp1, an essential RNA polymerase II CTD phosphatase. Mol Cell 32:478-90

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