Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phosphoSer2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4-or phospho-Ser2-bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2-modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.ach stage of transcription relies on ordered recruitment and exchange of specific protein complexes that act on RNA polymerase II, its nascent transcripts, and the underlying chromatin. This dynamic process is orchestrated via patterned posttranslational modifications of the carboxyl-terminal domain (CTD). This unusual and essential domain of Rpb1, the largest component of the 12-subunit polymerase, consists of repeating Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptides (26 repeats in budding yeast and 52 in humans) (1). The mechanistic consequences of phosphorylating Ser5 and Ser2 have been well documented (2-11). However, the role of Thr4 phosphorylation (pThr4), and even the necessity of Thr4 for cellular survival, appears to differ among closely related species and between growth conditions within a given species (12-15). Recent mass spectrometric analysis of an extensively engineered CTD revealed a paucity of pThr4, raising questions about the importance of this mark (16). In contrast, similar studies found pThr4 marks at levels comparable to or greater than the ubiquitously placed pSer2 mark in both yeast and human cells (17). Thus, much remains to be understood about the natural abundance and functional role of pThr4 marks on the endogenous, unmodified CTD.Previous studies suggest that pThr4 has roles in transcriptional elongation, 3ʹ-...

Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination

“…All these termination factors can bind Thr4P in vitro ( Table S1 ). In agreement with recent studies ( Harlen et al., 2016 , Jasnovidova et al., 2017 , Nemec et al., 2017 ), Rtt103 (Rhn1 in S. pombe ) interacts equally well with Ser2P and Thr4P ( Figure S4 D; Table S1 ). All proteins show no binding to Tyr1P in support of the proposed role for this residue in negatively affecting recruitment of termination factors ( Mayer et al., 2012 ).…”

RNA polymerase II CTD interactome with 3′ processing and termination factors in fission yeast and its impact on phosphate homeostasis

“…These results would place Thr4-PO 4 and Rhn1 in the same subpathway of termination in fission yeast. Our data are consistent with recent studies in budding yeast that establish a physical association of Thr4-phosphorylated CTD with Rtt103 (35,36,38), although in budding yeast the T4A mutation impacted termination specifically at small nucleolar RNA genes (35). A budding yeast T4V mutant strain exhibited a more general increase in Pol2 density distal to poly(A) sites of protein-coding genes (36).…”

“…Rtt103 is a "reader" of the Thr4-PO 4 and Ser2-PO 4 letters of the CTD code. Structures of the Rtt103 CTD interaction domain in complex with CTD-Thr4-PO 4 and CTD-Ser2-PO 4 peptides have delineated the determinants of CTD recognition (34)(35)(36)(37), as depicted in Fig. 7A for the Rtt103 complex with the CTD peptide PSY 1 S 2 P 3 (pT 4 )S 5 P 6 S 7 YSPTSPS containing a single Thr4-PO 4 heptad and a downstream unmodified heptad (36).…”

The RNA polymerase II CTD “orphan” residues: Emerging insights into the functions of Tyr-1, Thr-4, and Ser-7

“…An additional study using a T4A mutant found that this termination defect also affects a subset of snoRNA genes, and this was distinct from a known Ser2P requirement. 54 Additionally, NMR studies revealed that the Rtt103 CTD interacting domain (CID) binds to a Thr4P CTD as well as a Ser2P CTD, but the presence of both together inhibits binding. 55 While no study suggests that Thr4 is necessary globally for transcription, there is some question as to how prevalent Thr4P is in yeast, with one estimate at only 2% of Ser2P levels 14 and another suggesting it is as abundant as Ser2P.…”