Steven West

Human 5′ → 3′ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites

Eukaryotic protein-encoding genes possess poly(A) signals that define the end of the messenger RNA and mediate downstream transcriptional termination by RNA polymerase II (Pol II). Termination could occur through an ‘anti-termination’ mechanism whereby elongation factors dissociate when the poly(A) signal is encountered, producing termination-competent Pol II. An alternative ‘torpedo’ model postulated that poly(A) site cleavage provides an unprotected RNA 5′ end that is degraded by 5′ → 3′ exonuclease activities (torpedoes) and so induces dissociation of Pol II from the DNA template. This model has been questioned because unprocessed transcripts read all the way to the site of transcriptional termination before upstream polyadenylation. However, nascent transcripts located 1 kilobase downstream of the human β-globin gene poly(A) signal are associated with a co-transcriptional cleavage (CoTC) activity that acts with the poly(A) signal to elicit efficient transcriptional termination. The CoTC sequence is an autocatalytic RNA structure that undergoes rapid self-cleavage. Here we show that CoTC acts as a precursor to termination by presenting a free RNA 5′ end that is recognized by the human 5′ → 3′ exonuclease Xrn2. Degradation of the downstream cleavage product by Xrn2 results in transcriptional termination, as envisaged in the torpedo model.

Pause Sites Promote Transcriptional Termination of Mammalian RNA Polymerase II

ABSTRACT Polymerase II (Pol II) transcriptional termination depends on two independent genetic elements: poly(A) signals and downstream terminator sequences. The latter may either promote cotranscriptional RNA cleavage or pause elongating Pol II. We demonstrate that the previously characterized MAZ4 pause element promotes Pol II termination downstream of a poly(A) signal, dependent on both the proximity of the pause site and poly(A) signal and the strength of the poly(A) signal. The 5′→3′ exonuclease Xrn2 facilitates this pause-dependent termination by degrading the 3′ product of poly(A) site cleavage. The human β-actin gene also possesses poly(A) site proximal pause sequences, which like MAZ4 are G rich and promote transcriptional termination. Xrn2 depletion causes an increase in both steady-state RNA and Pol II levels downstream of the β-actin poly(A) site. Taken together, we provide new insights into the mechanism of pause site-mediated termination and establish a general role for the 5′→3′ exonuclease Xrn2 in Pol II termination.

Molecular Dissection of Mammalian RNA Polymerase II Transcriptional Termination

Summary Transcriptional termination of mammalian RNA polymerase II (Pol II) is an essential but little-understood step in protein-coding gene expression. Mechanistically, termination by all DNA-dependent RNA polymerases can be reduced to two steps, namely release of the RNA transcript and release of the DNA template. Using a simple nuclear fractionation procedure, we have monitored transcript and template release in the context of both natural and artificial Pol II terminator sequences. We describe the timing and relationship between these events and in so doing establish the roles of the poly(A) signal, cotranscriptional RNA cleavage events, and 5′-3′ exonucleolytic RNA degradation in the mammalian Pol II termination process.

Transcriptional Termination Enhances Protein Expression in Human Cells

Summary Transcriptional termination of mammalian RNA polymerase II (Pol II) requires a poly(A) (pA) signal and, often, a downstream terminator sequence. Termination is triggered following recognition of the pA signal by Pol II and subsequent pre-mRNA cleavage, which occurs either at the pA site or in transcripts from terminator elements. Although this process has been extensively studied, it is generally considered inconsequential to the level of gene expression. However, our results demonstrate that termination acts as a driving force for optimal gene expression. We show that this effect is general but most dramatic where weak or noncanonical pA signals are present. We establish that termination of Pol II increases the efficiency of pre-mRNA processing that is completed posttranscriptionally. As such, transcripts escape from nuclear surveillance.

Co‐transcriptional degradation of aberrant pre‐mRNA by Xrn2

Eukaryotic protein‐coding genes are transcribed as pre‐mRNAs that are matured by capping, splicing and cleavage and polyadenylation. Although human pre‐mRNAs can be long and complex, containing multiple introns and many alternative processing sites, they are usually processed co‐transcriptionally. Mistakes during nuclear mRNA maturation could lead to potentially harmful transcripts that are important to eliminate. However, the processes of human pre‐mRNA degradation are not well characterised in the human nucleus. We have studied how aberrantly processed pre‐mRNAs are degraded and find a role for the 5′→3′ exonuclease, Xrn2. Xrn2 associates with and co‐transcriptionally degrades nascent β‐globin transcripts, mutated to inhibit splicing or 3′ end processing. Importantly, we provide evidence that many endogenous pre‐mRNAs are also co‐transcriptionally degraded by Xrn2 when their processing is inhibited by Spliceostatin A. Our data therefore establish a previously unknown function for Xrn2 and an important further aspect of pre‐mRNA metabolism that occurs co‐transcriptionally.

Human Pcf11 enhances degradation of RNA polymerase II-associated nascent RNA and transcriptional termination.

The poly(A) (pA) signal possesses a dual function in 3′ end processing of pre-mRNA and in transcriptional termination of RNA polymerase II (Pol II) for most eukaryotic protein-coding genes. A key protein factor in yeast and Drosophila Pol II transcriptional termination is the 3′-end processing factor, Pcf11. In vitro studies suggest that Pcf11 is capable of promoting the dissociation of Pol II elongation complexes from DNA. Moreover, several mutant alleles of yeast Pcf11 effect termination in vivo. However, functions of human Pcf11 (hPcf11) in Pol II termination have not been explored. Here we show that depletion of hPcf11 from HeLa cells reduces termination efficiency. Furthermore, we provide evidence that hPcf11 is required for the efficient degradation of the 3′ product of pA site cleavage. Finally, we show that these functions of hPcf11 require an intact pA signal.

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1D83G/D83G mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1D83G/D83G mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1−/−). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs

ABSTRACT Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3′-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.

Splicing-coupled 3′ end formation requires a terminal splice acceptor site, but not intron excision

Splicing of human pre-mRNA is reciprocally coupled to 3′ end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3′ end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3′ end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3′ end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3′ end formation, but that on-going intron removal is less critical.

The increasing functional repertoire of U1 snRNA

Splicing is a key process for mRNA maturation, particularly in higher eukaryotes where most protein-coding transcripts contain multiple introns. It is achieved by the concerted action of five snRNAs (small nuclear RNAs) and hundreds of accessory proteins that form the spliceosome. Although snRNAs are present in equal amounts in the spliceosome, there is an overall excess of U1 in human cells. This finding led to the opinion that U1 might be involved in processes other than splicing. Research has shown that this is indeed the case and some examples found from studies in human cell systems are described briefly in the present review.