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.
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.
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