Periodic Reporting for period 4 - DECOR (Dynamic assembly and exchange of RNA polymerase II CTD factors)
Período documentado: 2020-02-01 hasta 2020-07-31
The DECOR project focused on dynamic assembly and exchange of RNA polymerase II CTD factors. We performed the structural analysis of RNA polymerase II (Pol II) C-terminal domain (CTD) interactome using approaches of integrated structural biology. Pol II CTD associates with many regulatory factors in a weak and transient manner throughout the transcription cycle. The association is dictated by different post-translational modification patterns and conformational changes of the CTD. The mechanism of how these accessory factors assemble and exchange on the CTD of Pol II has remained a major challenge. The project had and achieved three major objectives: (i) To determine 3D structures of several protein factors bound to the modified CTD: We solved several structures of regulatory factors bound to the phosphorylated CTD. The structural data in conjunction with binding assays enabled us to decode how CTD modification patterns stimulate or prevent binding of a given regulatory factor. (ii) To understand how the overall CTD structure is remodeled by binding of multiple copies of processing factors and how these factors cross-talk with each other: We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3′-end RNA-processing and transcription termination factor. We found that CTD–Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins. (iii)To elucidate the mechanistic basis for exchange of processing factors on the CTD: We found that some regulatory factors contain sequences which can mimic the CTD motifs of Pol II. This opens up the exciting possibility that the dynamics of factors interacting with the CTD throughout the transcription cycle is regulated not only by the enzymes responsible for CTD modifications, but also by competitive interactions with proteins containing CTD-like motifs that mimic particular CTD-modification pattern. We demonstrated this exchange and competition mechanisms are used by Sen1-dependent termination and 3’-5’ (exosome−TRAMP4) or 5’−3’ (Rat1−Rai1) RNA degradation machineries coupled to Pol II CTD.
Objective 1:(i) We determined the solution structure of the Rtt103p CTD‐interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure revealed a direct recognition of the phospho‐Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Our structural data suggest that the recruitment of a CID‐containing CTD‐binding factor may be coded by more than one letter of the CTD code (doi: 10.15252/embr.201643723). (ii) We also investigated the interaction of transcription elongation factor Spt6 associates with Pol II CTD, which is mediated via a tandem SH2 (tSH2) domain. We reported that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Overall, our structural studies revealed a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers (doi: 10.1016/j.jmb.2020.05.007). (iii) To facilitate the above-mentioned study with Y1-phosphorylated CTD, we developed co-expression system for modification enzyme and SUMO-tagged intrinsically disordered proteins (IDPs) with a subsequent purification procedure that allows for the production of modified IDP (doi: 10.2144/btn-2019-0033). (iv) we were also searching for a new CTD binders using GST pull-downs in human cells and identified several proteins which contained a new CTD-binding domain, SPOC. One of the protein containing SPOC is PHD-finger protein 3 (PHF3) and it preferentially binds S2-phosphorylated CTD. In a collaboration with Dea Slade, we conducted an interdisciplinary study which showed that PHF3 tracks with Pol II across the length of genes and negatively regulates transcription and mRNA stability (Appel LM, et al. PHF3 regulates neuronal gene expression through the new Pol II CTD reader domain SPOC, submitted). Objective 2: We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3′-end RNA-processing and transcription termination factor. The CTD–Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins (doi: 10.1073/pnas.1712450114). Objective 3: (i) We identified a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy‐terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD‐interacting domain of Nrd1, an RNA‐binding protein that binds specific sequences in ncRNAs. In combination with functional assays, the data shed light on the network of protein–protein interactions that control termination of non‐coding transcription by Sen1 (doi: 10.15252/embj.2019101548). (ii) We identified motifs in an intrinsically disordered region of Trf4 (subunit of TRAMP complex) and Rat1-Rai1 complex that mimic the phosphorylated pol II CTD. We uncovered the involvement of CID-containing Rtt103 that connects 3’-5’ (exosome−TRAMP4) or 5’−3’ (Rat1−Rai1) RNA degradation machineries with Pol II in order to dismantle transcribing RNAP II from DNA template (Kabzinski et al. Rtt103 as an adaptor connecting 3’-to-5’ and 5’-to-3’ RNA degradation machineries with RNA polymerase II. Manuscript in preparation). The project has opened a new research direction in regard to the crosstalk between Pol II transcription and DNA damage response mechanisms. Furthermore, we continue to explore how regulatory factors that associate with Pol II CTD promote liquid-liquid phase separation of RNA polymerase II, an important phenomenon that dynamically control partitioning of transcription machinery in the nucleus. This line of research has therapeutic potential, as we show that cancer-associated mutations in some genes (e.g. BRCA1) alter the ability of Pol II to undergo phase-separation to form membrane-less organelles (Sebesta M, et al. CTD binders promote phase separation of phosphorylated RNA Polymerase II CTD. manuscript in preparation). Taken together, the scientific aims of the project were completed despite the project faced a number of common unpredictable issues, which always appear in projects of this kind. The work on publication of the remaining data is in progress and will be completed in due time.
The project progressed beyond the state-of-the-art thanks to a combination of several structural biology approaches and advanced expression systems in insect cells. We also found that regulatory factors that associate with Pol II CTD promote liquid-liquid phase separation of RNA polymerase II, an important phenomenon that dynamically control partitioning of transcription machinery in the nucleus.