The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol2) consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The CTD is essential for cell viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation. The inherently plastic CTD structure is modulated by dynamic phosphorylation and dephosphorylation of the heptad serine (S2, S5, S7), threonine (T4), and tyrosine (Y1) residues. The phospho-status of the CTD provides informational cues about the transcription machinery - a CTD code - that is read by CTD receptor proteins. Our goals in this project are to understand how CTD information is inscribed, organized, and transduced to cellular effectors, and how the CTD code governs gene expression. We are deciphering the code by genetically manipulating the composition and structure of the fission yeast Pol2 CTD. This approach has taught us that: (i) the Ser2, Thr4, and Ser7 phospho-sites are not essential for fission yeast viability; (ii) Phe is functional in lieu of Tyr1; and (iii) Ser5 is the only strctly essential phosphorylation mark, inscription of which requires Pro6. We've shown that the chief function of the Ser5-PO4 mark is to recruit the mRNA capping apparatus to nascent Pol2 transcripts. The outstanding challenge now is to understand how CTD coding letters are assembled into words (i.e., a vocabulary). This project aims to define the physiology of individual coding letters, and the rudiments of a CTD vocabulary, via two complementary approaches. To gauge the output of the code, we will perform transcriptome profiling of our collection of fission yeast CTD mutants. To gain new insights to steps in gene expression that rely on particular CTD cues, we will analyze mutational synergies of Pol2 CTD mutants (e.g., synthetic lethal interactions) and their allele-specificities. Initial results highlight that the fssion yeast phosphate and iron homeostasis regulons, which are controlled by specific DNA-binding transcription factors, are strongly influenced by mutation of individual CTD marks. We will dissect biochemically and structurally the Pho7 and Fep1 transcription factors that control the phosphate and iron regulons, and probe their functional/physical interactions with the CTD. Finally, we will illuminate the structure, mechanism, and specificity of the essential fission yeas CTD phosphatase Fcp1.

Public Health Relevance

CTD phosphorylation dynamics orchestrate Pol2 transcription and co-transcriptional RNA processing. The phospho-CTD code is deeply rooted in eukaryal biology and our studies of how CTD information is inscribed, organized, and transduced in the fission yeast S. pombe will illuminate core principles that are applicable broadly. Our investigations of fission yeast Fcp1 as a paradigmatic CTD phosphatase will inform the connection of Fcp1 to human pathology, whereby a partial deficiency of human Fcp1 is associated with an autosomal recessive developmental disorder characterized by cataracts, facial dysmorphism, and peripheral neuropathy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1)
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Bender, Michael T
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Sloan-Kettering Institute for Cancer Research
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Garg, Angad; Sanchez, Ana M; Shuman, Stewart et al. (2018) A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking prt lncRNA and pho1 genes. J Biol Chem 293:4456-4467
Garg, Angad; Goldgur, Yehuda; Schwer, Beate et al. (2018) Distinctive structural basis for DNA recognition by the fission yeast Zn2Cys6 transcription factor Pho7 and its role in phosphate homeostasis. Nucleic Acids Res 46:11262-11273
Roth, Allen J; Shuman, Stewart; Schwer, Beate (2018) Defining essential elements and genetic interactions of the yeast Lsm2-8 ring and demonstration that essentiality of Lsm2-8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24. RNA 24:853-864
Sanchez, Ana M; Shuman, Stewart; Schwer, Beate (2018) Poly(A) site choice and Pol2 CTD Serine-5 status govern lncRNA control of phosphate-responsive tgp1 gene expression in fission yeast. RNA 24:237-250
Sanchez, Ana M; Shuman, Stewart; Schwer, Beate (2018) RNA polymerase II CTD interactome with 3' processing and termination factors in fission yeast and its impact on phosphate homeostasis. Proc Natl Acad Sci U S A 115:E10652-E10661
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

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