Review Article
Simplicity is the Ultimate Sophistication—Crosstalk of Post-translational Modifications on the RNA Polymerase II

https://doi.org/10.1016/j.jmb.2021.166912Get rights and content

Highlights

  • The largest subunit of RNA Pol II is heavily post-translational modified during transcription.

  • These modifications now can be accurately mapped by mass spectrometry.

  • The crosstalk among residue modifications results in differentiated transcriptional outcomes.

  • The Pol II modifications communicate with histones to ensure smooth transcription progress.

Abstract

The highly conserved C-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises a consensus heptad (Y1S2P3T4S5P6S7) repeated multiple times. Despite the simplicity of its sequence, the essential CTD domain orchestrates eukaryotic transcription and co-transcriptional processes, including transcription initiation, elongation, and termination, and mRNA processing. These distinct facets of the transcription cycle rely on specific post-translational modifications (PTM) of the CTD, in which five out of the seven residues in the heptad repeat are subject to phosphorylation. A hypothesis termed the “CTD code” has been proposed in which these PTMs and their combinations generate a sophisticated landscape for spatiotemporal recruitment of transcription regulators to Pol II. In this review, we summarize the recent experimental evidence understanding the biological role of the CTD, implicating a context-dependent theme that significantly enhances the ability of accurate transcription by RNA polymerase II. Furthermore, feedback communication between the CTD and histone modifications coordinates chromatin states with RNA polymerase II-mediated transcription, ensuring the effective and accurate conversion of information into cellular responses.

Introduction

RNA polymerase II (Pol II) is a multi-subunit enzyme involved in transcribing all protein-coding genes in eukaryotes.1, 2 The largest subunit of Pol II (RPB1) contains a series of seven residue (Heptad) repeats as its Carboxyl-Terminal domain (CTD). The CTD interacts with various general and specific transcription factors, regulators, and other proteins to aid and regulate transcription.3, 4, 5 Across eukaryotes, the number of repeats and the level of conservation differ among species. For example, Saccharomyces cerevisiae contains 26 repeats, which mostly conform to the consensus sequence of Tyrosine (Tyr1), Serine (Ser2), Proline (Pro3), Threonine (Thr4), Serine (Ser5), Proline (Pro6), and Serine (Ser7)3 (Figure 1(a) and (b)). The positioning of heptads can be viewed differently with alternative frames of references though (Figure 1(c)). Through the evolution tree, complicated eukaryotes contain Pol II with the CTD heptads deviating from the consensus, mostly in the 7th position6 (Figure 1(b)). The most extreme example is Drosophila, which has a CTD consisting of 42 repeats of highly divergent sequence with only two heptads as consensus.7 Even though the CTD doesn't directly contribute to Pol II's catalytic activity, its function is required for the effective transcription of genetic information cells with the CTD deleted cannot survive.8, 9

The CTD heptad repeats undergo extensive post-translational modifications (PTMs) such as phosphorylation,10 acetylation,11 methylation,12 and glycosylation.13 Phosphorylation is the most prominent modification in which kinases phosphorylate Ser2 and Ser5 of the heptad transiently. They are returned to their dephosphorylated form by the end of the cycle14 (Figure 1(a)). When RNA Pol II binds to the promoter, it is free of phosphate modifications. Ser5 is among the first residues to be phosphorylated after transcriptional initiation.15 After initiation, the DRB-sensitivity inducing factor (DSIF) and the negative elongation factor (NELF) block Pol II at ~60 bp downstream of the transcription start site, called promoter-proximal pausing.16, 17 Pausing is released by the positive transcription elongation factor (P-TEFb) when it phosphorylates negative elongation factors (DSIF and NELF), as well as Ser2 of the CTD.18 These phosphorylation marks are removed at the end of the transcriptional cycle, which prepares Pol II to initiate another transcription cycle.

Spatiotemporal phosphorylation of the CTD is critical for Pol II's biological function since mutations on the residues of heptads or the enzymes modifying the phosphorylation states compromise transcriptional functions.19, 20 The high abundance of PTM sites on the flexible CTD exhibits resemblance to the flexible histone tails that are also heavily modified by methylation, acetylation, ubiquitination, and phosphorylation.21, 22, 23 In 2003, a CTD code hypothesis was proposed, highlighting its potential to encode a large amount of information through its PTMs and their combinations to recruit binding partners.24 In a similar fashion to proteins interacting with histones, the proteins interacting with the CTD are identified as writers (such as kinases that place phosphate groups), modifiers (such as proline isomerases that change proline isomeric states), readers (such as proteins physically recruited to Pol II by interacting with the CTD), and erasers (such as phosphatases that remove phosphates) (Table 1). The interplay between this extensive collection of writers/modifiers/readers/erasers can generate all combinations of modification species, leading to various functions during transcription (Figure 1(d) and (e)). It has been twenty years since the CTD code was proposed, yet the exact mechanism of CTD-mediated transcription still evades our understanding.

Section snippets

Detection of CTD Phosphorylation

The combinatorial coding system is highly attractive, with great potential for information encryption. However, experimental evidence which supports the combinatorial PTMs of CTD has been elusive due to the lack of adequate tools for detecting and identifying these chemical modifications. Historically, the detection of phosphorylation species can be achieved using electrophoretic mobility assays.25, 26 While fast and robust, these assays are unable to identify the specific phosphorylated

Crosstalk of Different Residues on the CTD Heptad

The histone modification hypothesis describes the generation of sophisticated PTM patterns for precise transcriptional control, achieved by the crosstalk between different histone PTMs. For example, Histone-2-B Lysine-120 ubiquitination (H2BK120ub) regulates the methylation of H3K79 and H3K4, which activates gene expression.43, 44 In turn, the tri-methylation of H3K4me3 and H3K36me3 both inhibit the Protein Regulator of Cytokinesis (PRC) 2 activity, ensuring gene expression.45 In contrast,

Crosstalk Between RNA Polymerase II and Histone

Histones control the compactness and accessibility of DNA, which is key to Pol II's function in transcription. Crosstalks between histones and Pol II ensures that transcription can progress efficiently without delay. Communication with histones is likely to be one of the CTD’s essential functions since in vitro reconstruction shows that a transcript can be produced without the CTD modifications.88, 89, 90 Yet, the loss of the CTD in cells is fatal.19 One reasonable explanation is that the CTD

Perspective

The C-terminal domain of the largest subunit of Pol II is enriched with sites for post-translational modification. In combination with the existence of divergent heptads, there is a massive potential for variation. Crosstalk between modifications on both consensus and divergent sequences leads to a staggering capacity for transmitting information, which may ultimately alter transcription outcomes. By developing mass spectrometry techniques, we can more accurately identify the positions of

CRediT authorship contribution statement

Mukesh Kumar Venkat Ramani: Conceptualization, Visualization, Writing - original draft. Wanjie Yang: Visualization. Seema Irani: Visualization. Yan Zhang: Conceptualization, Writing - original draft.

Acknowledgment

We are grateful for Dr. Blasé LeBlanc’s critiques on our manuscript. We thank the National Institutes of Health (R01GM104896 and R01GM125882 to Y.J.Z) and Welch Foundation (F-1778 to Y.Z.) for supporting our research.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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