Review
Ubiquitylation and degradation of elongating RNA polymerase II: The last resort

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Abstract

During its journey across a gene, RNA polymerase II has to contend with a number of obstacles to its progression, including nucleosomes, DNA-binding proteins, DNA damage, and sequences that are intrinsically difficult to transcribe. Not surprisingly, a large number of elongation factors have evolved to ensure that transcription stalling or arrest does not occur. If, however, the polymerase cannot be restarted, it becomes poly-ubiquitylated and degraded by the proteasome. This process is highly regulated, ensuring that only RNAPII molecules that cannot otherwise be salvaged are degraded. In this review, we describe the mechanisms and factors responsible for the last resort mechanism of transcriptional elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Highlights

► Ubiquitylation and degradation of RNA polymerase II is a last resort pathway. ► Ubiquitylation occurs via a multi-step mechanism involving two distinct E3s and proofreading by ubiquitin proteases. ► The stopped form of RNA polymerase II is specifically targeted.

Introduction

RNA polymerase II (RNAPII) transcript elongation is not a smooth, continuous process. Rather, the polymerase frequently stalls, and even backtracks, leading to transcriptional arrest. Many factors contribute to the processivity of RNAPII, helping to deal with transitory pauses and stalls [1]. Conditions leading to stalling can be in cis, such as sequences that are difficult to transcribe [2], [3], [4], topological constraints [5], [6], [7], [8], or chromatin structure [9], [10], [11], or in trans, such as depletion of NTPs required for RNA polymerization [12]. Temporary stalls can lead to a backtracked RNAPII and transcriptional arrest, and this can often be dealt with by the recruitment of elongation factors such as TFIIS [1]. Other causes of transcriptional arrest include bulky DNA adducts, which block the path of transcribing RNAPII [13], [14], [15]. The great stability of the RNAPII elongation complex allows the polymerase to remain attached to its template rather than dissociating from sites of disruption. Such elongation complex stability is a blessing, ensuring high fidelity of long transcripts, but also a curse when polymerase stops its progression, as all transcription of the affected gene is now blocked.

RNAPII stalled at helix-distorting DNA damage is doubly dangerous, because not only is the gene not transcribed, but the polymerase also blocks access of the nucleotide excision repair factors to the lesion [13]. Not surprisingly, cells have evolved multiple ways to deal with such persistently stopped RNAPII. For example, the polymerase itself acts as a DNA damage sensor, leading to more rapid repair of the transcribed strand (TS) of a gene compared to the non-transcribed strand (NTS) or the genome overall [14], [15]. Most DNA damage encountered by RNAPII is dealt with by a dedicated transcription-coupled nucleotide excision repair (TC-NER) pathway. In Saccharomyces cerevisiae, TC-NER is mediated by Rad26, and in mammals by Cockayne syndrome proteins A and B (CSA and CSB). The TC-NER pathway has been extensively studied and is described elsewhere (see review by Gaillard and Aguilera in this issue).

When the TC-NER pathway is unable to allow continued transcription, an alternative pathway is required to remove the stalled RNAPII and allow repair factors access to the lesion. One proposed mechanism is for RNAPII to bypass the bulky lesions, but this process is both slow and extremely inefficient [16], [17]. Transcription-blocking DNA damage can also result in the poly-ubiquitylation and degradation of the largest subunit of RNAPII, Rpb1 [18], [19], [20], [21]. Such removal would allow repair of the lesion to occur. Initially, it was hypothesized that RNAPII removal was required for normal TC-NER: Rpb1 was thought to be ubiquitylated and removed from sites of damage to allow access for TC-NER factors [18]. However, subsequent work has shown that the RNAPII degradation pathway is instead an entirely separate, more drastic “last resort” pathway, which acts as an alternative to TC-NER [21], [22]. Accordingly, TC-NER impairment actually leads to increased Rpb1 poly-ubiquitylation and degradation [21], [23], [24].

It is important to stress that although it was originally identified as a response to transcription-obstructing DNA damage, Rpb1 poly-ubiquitylation and degradation occurs under a number of conditions that lead to persistent transcriptional stalling/arrest [22], [25], [26], [76]. For example, treatment of cells with the elongation inhibitor α-amanitin [20], [22], or mutation of the gene encoding transcript cleavage factor TFIIS [25], also results in Rpb1 ubiquitylation and degradation. Since the initial discovery of the “last resort” pathway, it has proven to be much more complex than originally thought.

In general, protein ubiquitylation was originally discovered as a signal for the degradation of proteins, but it has later emerged that it is a multi-functional post-translational modification with many distinct outcomes [27], [28]. The substrate of ubiquitylation becomes conjugated on an internal lysine, via a triple enzyme cascade, with ubiquitin being transferred from an E1 (activating) enzyme, to an E2 (conjugating) enzyme and finally, via the help of an E3 (ligating) enzyme, to the substrate. Ubiquitin itself can be extended to create chains of differing topology and promoting different outcomes, with only lysine (Lys)-48 linked chains targeting proteins directly to the proteasome for degradation. In the remainder of this review we will outline the current model for the RNAPII ubiquitylation pathway, focusing on the mechanism of Rpb1 recognition and degradation for gene clearance and the continuation of transcription.

Section snippets

A multi-step mechanism for RNAPII ubiquitylation and degradation

Over the last decade it has become evident that, as might be expected for a crucial cellular protein, ubiquitylation and degradation of RNAPII is highly regulated. It occurs via a multi-step process. In the initial step, the polymerase is mono-ubiquitylated by one ubiquitin ligase (E3), before another takes over to perform poly-ubiquitylation. Both these steps can be “proof-read” and reversed by specific ubiquitin proteases. Poly-ubiquitylation then attracts proteins that help guide the

Recognition of stalled RNAPII

To avoid undesirable side-effects on general cellular transcription, it is obviously critical that ubiquitylation and degradation of Rpb1 is confined to RNAPII molecules that are arrested. Apart from the proofreading mechanisms outlined above, permanently stalled RNAPII must thus represent a substrate that the ubiquitylation system can distinguish from normal, actively transcribing RNAPII, and from free RNAPII. The precise manner in which this is achieved remains unclear though some likely

Choice of RNAPII degradation over TC-NER/restart

As mentioned above, it is imperative that degradation of Rpb1 acts as a last resort. Intuitively, the “restart” and TC-NER machineries must first “sample” the stalled RNAPII, with degradation the last remaining option if these other pathways fail. Accordingly, mechanisms may have evolved to ensure that there is a difference in the swiftness of transcription restart, TC-NER, and degradation of Rpb1. For example, elongation factors that increase the processivity of transcript elongation may

Concluding remarks

Recent studies have revealed that the poly-ubiquitylation of Rpb1 in response to permanent stalling or arrest is a two‐step, “last resort” pathway, with many factors involved. While we understand the basic mechanism of Rpb1 degradation (see Fig. 1), there are many questions left unanswered. This review highlights some of the pertinent questions that focus on individual steps in the pathway. More generally, we also need to understand how the pathway is regulated by dynamic compartmentalization

Acknowledgements

This work was supported by grants from Cancer Research UK and European Research Council (ERC) (to J.Q.S.). Members of the Svejstrup laboratory are thanked for helpful discussions and comments on the article.

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