Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative ...polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
Display omitted
•Human SCAF4 and SCAF8 couple RNAPII Ser2P- and Ser5P-binding and RNA processing•SCAF4 and SCAF8 bind nascent RNA upstream of early polyadenylation sites•SCAF4 and SCAF8 prevent early mRNA transcript cleavage and polyadenylation•Lack of SCAF4 and SCAF8 result in truncated protein products and is cell lethal
Eukaryotic anti-terminator proteins suppress usage of early polyadenylation sites to prevent production of truncated proteins.
In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were ...performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.
Display omitted
•A multiomic screening approach examines the UV-induced DNA damage response•Multiple factors are connected to the transcription-related DNA damage response•Melanoma gene STK19 is required for a normal DNA damage response
Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered.
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.
► 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.
The multi-subunit Elongator complex was first identified by its association with an RNA polymerase II holoenzyme engaged in transcriptional elongation, and subsequent data have provided further ...evidence that the complex is involved in histone acetylation and transcription. However, most Elongator is cytoplasmic, and recent data has indicated a role in processes as diverse as exocytosis and tRNA modification. One of the subunits of Elongator is encoded by a gene that is mutated in patients suffering from the severe neurodevelopmental disorder familial dysautonomia.
Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to ...transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle; this is the functional form of the nascent pre-mRNA and determines the fate of the mature transcript. However, factors that connect the transcribing polymerase with the mRNP particle and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD)) as subunits of a novel protein complex--named DBIRD--that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.
During transcript elongation in vitro, backtracking of RNA polymerase II (RNAPII) is a frequent occurrence that can lead to transcriptional arrest. The polymerase active site can cleave the ...transcript during such backtracking, allowing transcription to resume. Transcript cleavage is either stimulated by elongation factor TFIIS or occurs much more slowly in its absence. However, whether backtracking actually occurs in vivo, and whether transcript cleavage is important to escape it, has been unclear. Using a yeast TFIIS mutant that lacks transcript cleavage stimulatory activity and simultaneously inhibits unstimulated cleavage, we now provide evidence that escape from backtracking via transcript cleavage is essential for cell viability and efficient transcript elongation. Our results suggest that transcription problems leading to backtracking are frequent in vivo and that reactivation of backtracked RNAPII is crucial for transcription.
Display omitted
► A TFIIS mutant that inhibits intrinsic RNAPII transcript cleavage has been isolated ► Transcript cleavage by RNAPII is essential for transcription and viability ► RNAPII backtracking must be a frequent event in vivo ► Irreversibly backtracked RNAPII is a target for ubiquitylation/degradation
RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA ...polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast
Saccharomyces cerevisiae,
which does not have a
RECQL5
ortholog. We found that
RECQL5
expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in
S. cerevisiae
.
Transcript Elongation by RNA Polymerase II Selth, Luke A; Sigurdsson, Stefan; Svejstrup, Jesper Q
Annual review of biochemistry,
01/2010, Volume:
79, Issue:
1
Journal Article
Peer reviewed
Until recently, it was generally assumed that essentially all regulation of transcription takes place via regions adjacent to the coding region of a gene--namely promoters and enhancers--and that, ...after recruitment to the promoter, the polymerase simply behaves like a machine, quickly "reading the gene." However, over the past decade a revolution in this thinking has occurred, culminating in the idea that transcript elongation is extremely complex and highly regulated and, moreover, that this process significantly affects both the organization and integrity of the genome. This review addresses basic aspects of transcript elongation by RNA polymerase II (RNAPII) and how it relates to other DNA-related processes.
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction ...of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Display omitted
•UV elicits elongation slowdown and restricts transcription to the 5′ end of genes•UV induces a switch from long to short alternative last exon (ALE) transcript isoforms•ASCC3 short and long ALE isoforms have antagonistic functions in the UV response•The UV-induced ASCC3 short isoform functions as a long non-coding RNA
UV damage generates a functional non-coding RNA through alternative pre-mRNA processing of a damage response factor transcript, identifying a pathway for repurposing protein coding genes under selective conditions.
PDF
