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 Y₁S₂P₃T₄S₅P₆S₇ 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 phospho-Ser2 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.

Keywords: CTD code; CTD interactome; NMR; noncoding RNA; phosphothreonine.

Fig. 1.

pThr4 levels correlate with snoRNA termination in S. cerevisiae. (A) Rpb1 with mutant CTDs are shown with black (WT), orange (T4A), and blue (T4E) CTD repeats. Plasmids bearing mutant Rpb1 and a LEU2 auxotrophic marker were transformed into yeast. Shown are serial dilutions of cells in the presence of 5-FOA, which counterselects against yeast retaining the URA3-bearing plasmid containing WT Rpb1. −LEU, agar plates lacked leucine; OD, optical density; −URA, agar plates lacked uracil. (B) T4A expression (from genome tiling microarrays) normalized to WT is plotted across the whole yeast genome with chromosomes (Chr.) listed below. Transcripts above the red line denote regions with z ≥ 5 (*protein-coding genes). (C) RNA expression (Top) and Rpb3 ChIP analysis (Bottom) from WT (black), T4A (orange), or S2A/WT (green) are shown at SNR33. Arrows denote the transcription start site, black bars indicate gene bodies, and red lines indicate cleavage and polyadenylation sites. Northern blot shows probing SNR33 (Right). IP, immunoprecipiate. SCR1 is a loading control. (D) Same as C at the SNR40 locus.

Fig. 2.

Thr4-dependent and -independent snoRNAs. (A) The 3′-extension index (EI) for each snoRNA in T4A is plotted from highest to lowest. Orange bars indicate an EI of >1.7. (B) Example gene (SNR13) and equation used to calculate EI, defined as fold change in the average expression 200 bp downstream of the 3′ end of snoRNAs. (C) Comparison of RNA extension index to Rpb3 ChIP read-through, defined as fold change in the average Rpb3 ChIP occupancy in the 1,000-bp window downstream of the 3′ end of snoRNAs. (D) Four snoRNA classes are illustrated with black boxes representing snoRNAs and purple representing exons. The fractions of snoRNAs in each class with 3′ extensions in T4A are noted next to the red dotted line depicting the mature 3′ end of respective snoRNAs. (E) pThr4 occupancy (normalized to Rpb3) centered at the 3′ end of SNR33 or SNR17A (Left) or a gene compilation of pThr4 occupancy across all snoRNAs read-through in T4A (dark) or no read-through (light). Shaded regions indicate 95% confidence interval. (F) Similar traces for pSer2.

Fig. 3.

Thr4 is required for association of elongation and termination factors. (A) Flowchart of the method to identify factors whose binding is compromised in T4A. (B) Volcano plot illustrating the fold change ln(T4A/WT) and the significance –Log₁₀[false discovery rate (FDR)] of differential binding. Proteins depleted at least one natural log in T4A with an FDR of <0.05 are color coded and labeled as in C. (C) Physical interconnectivity (46) between the significantly depleted proteins identified in B. Gray background indicates low density connectivity. (D) Flowchart of the experimental design to assay binding of factors to human CTD (Top). Phosphorylation state of HeLa extract, unphosphorylated human Rpb1, or Rpb1 phosphorylated with human PLK3 (Left). Enriched hypophosphorylated Pol II (Thr4-CTD) and PLK3-treated CTD (pThr4-CTD) was incubated with the indicated TAP-tagged proteins, expressed in yeast. Indicated proteins were assayed for binding to pThr4-bearing Rpb1 and probed with α-TAP (Right).

Fig. 4.

The CID domain of Rtt103 binds analogously to pThr4_ab-CTD and pSer2_ab-CTD peptides. (A) Schematic of diheptad CTD peptides used in NMR titrations. Each CTD repeat (arrow) is labeled as repeat “a” or repeat “b.” Sequences of the peptides are shown on the Top. Phosphothreonines and phosphoserines are indicated by orange and green circles, respectively. (B) Overlay of ¹H-¹⁵N HSQC spectra of free Rtt103-CID (black) and of the same domain in complex with pThr4_ab-CTD (orange) and pSer2_ab-CTD (green). (C) Chemical shift perturbations (CSPs) of Rtt103-CID with diheptad peptides phosphorylated at Thr4 (orange) or Ser2 (green). Very similar trends are observed for both peptides, with generally smaller overall amplitude changes for pThr4_ab-CTD (Top). (Bottom) Chemical shift difference for Rtt103-CID in complex with the two peptides. Three residues V109, I112, and K114 (red) have the largest chemical shift differences (>0.08 ppm) in the two complexes. (D) The secondary structure is unchanged for Rtt103-CID in complex with pThr4_ab-CTD. Chemical shift deviations from random coil shifts for ¹³Cα (Top), ¹³Cβ (Middle), and ¹³C′ (Bottom) versus residue number are shown. (E) Model of Rtt103-CID in complex with pThr4_ab-CTD, generated from the existing structure of Rtt103-pSer2_ab-CTD, by substituting pSer2 with Ser2 and Thr4 with pThr4 and performing energy minimization within PyMOL. The three amino acids experiencing the largest changes between the two phosphorylated peptides, V109, I112, and K114 (red), are located in the immediate proximity of the sites of phosphorylation (Ser2 or Thr4). (F) Affinities of Rtt103-CID for diheptad repeat CTD peptides, as measured by NMR titrations. Values are averaged over multiple amino acids (>10) used for extracting independent K_d values by fitting of chemical shift changes vs. the ratio of peptide to Rtt103. Data are represented as mean ± SD.

Fig. 5.

Model of Rtt103 in complex with pThr4-CTD. (A) Two orthogonal views of the model of Rtt103 in complex with pThr4-CTD and (B) representative solution state structures of Rtt103 in complex with pSer2-CTD are shown. All structural figures were generated with PyMOL (56). (C) Biochemical validation of the model is shown. Purified Rtt103 (WT) or the mutant Rtt103 (R108N) was incubated with a bead only control, or beads bound to GST-CTD that were either in their unphosphorylated state (Thr4-CTD) or phosphorylated state (pThr4-CTD). Bar graph shows fold change in binding to the phosphorylated CTD vs. unphosphorylated (*P < 0.01).