In response to transcription-blocking DNA lesions such as those generated by UV irradiation, cells activate a multipronged DNA damage response. This response encompasses repair of the lesions that ...stall RNA polymerase (RNAP) but also a poorly understood, genome-wide shutdown of transcription, even of genes that are not damaged. Over the past few years, a number of new results have shed light on this intriguing DNA damage response at the structural, biochemical, cell biological, and systems biology level. In this review we summarize the most important findings.
Biochemical and genetic studies indicate crosstalk between TC-NER factors and factors regulating RNAPII backtracking and elongation.
TC-NER is controlled by an unexpectedly complex layer of post-translational regulation targeting repair factors and RNAPII itself.
Dramatic changes to the transcriptional program in response to UV irradiation have been revealed by high-throughput genome-wide and time-resolved measurements.
Chromatin remodelers, splicing factors, noncoding RNAs, and RNA modifications provide a multifaceted transcriptional response to UV irradiation.
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5′ to 3′ ...in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3′ UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5′ to 3′ on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3′ UTRs.
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•MOV10 has in vitro 5′ to 3′ directional RNA unwinding activity•MOV10 displays widespread binding to 3′ untranslated regions (3′ UTRs) of mRNAs•PAR-CLIP of helicase-deficient MOV10 indicates impaired translocation along 3′ UTRs•MOV10 interacts with UPF1 and contributes to degradation of UPF1-regulated transcripts
RNA helicases regulate gene expression by remodeling RNA secondary structures and RNA-protein interactions. Gregersen et al. show that the RNA helicase MOV10 binds to 3′ UTRs and translocates 5′ to 3′ on its mRNA targets. MOV10 interacts with UPF1 and functions in UPF1-mediated mRNA degradation as mRNPase clearance factor.
MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its ...targets in colon cancer remains limited.
To investigate the role of miR-145 in colon cancer, we have employed a microarray based approach to identify miR-145 targets. Based on seed site enrichment analyses and unbiased word analyses, we found a significant enrichment of miRNA binding sites in the 3'-untranslated regions (UTRs) of transcripts down-regulated upon miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets.
The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor-suppressive functions of miR-145.
MicroRNAs (miRNAs) are well recognized as gene regulators and have been implicated in the regulation of development as well as human diseases. miR-143 is located at a fragile site on chromosome 5 ...frequently deleted in cancer, and has been reported to be down-regulated in several cancers including colon cancer.
To gain insight into the role of miR-143 in colon cancer, we used a microarray-based approach in combination with seed site enrichment analysis to identify miR-143 targets.
As expected, transcripts down-regulated upon miR-143 overexpression had a significant enrichment of miR-143 seed sites in their 3'UTRs. Here we report the identification of Hexokinase 2 (HK2) as a direct target of miR-143. We show that re-introduction of miR-143 in the colon cancer cell line DLD-1 results in a decreased lactate secretion.
We have identified and validated HK2 as a miR-143 target. Furthermore, our results indicate that miR-143 mediated down-regulation of HK2 affects glucose metabolism in colon cancer cells. We hypothesize that loss of miR-143-mediated repression of HK2 can promote glucose metabolism in cancer cells, contributing to the shift towards aerobic glycolysis observed in many tumors.
Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. ...We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.
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.
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•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.
Green sulfur bacteria (GSB) constitute a closely related group of photoautotrophic and thiotrophic bacteria with limited phenotypic variation. They typically oxidize sulfide and thiosulfate to ...sulfate with sulfur globules as an intermediate. Based on genome sequence information from 15 strains, the distribution and phylogeny of enzymes involved in their oxidative sulfur metabolism was investigated. At least one homolog of sulfide:quinone oxidoreductase (SQR) is present in all strains. In all sulfur-oxidizing GSB strains except the earliest diverging Chloroherpeton thalassium, the sulfide oxidation product is further oxidized to sulfite by the dissimilatory sulfite reductase (DSR) system. This system consists of components horizontally acquired partly from sulfide-oxidizing and partly from sulfate-reducing bacteria. Depending on the strain, the sulfite is probably oxidized to sulfate by one of two different mechanisms that have different evolutionary origins: adenosine-5'-phosphosulfate reductase or polysulfide reductase-like complex 3. Thiosulfate utilization by the SOX system in GSB has apparently been acquired horizontally from Proteobacteria. SoxCD does not occur in GSB, and its function in sulfate formation in other bacteria has been replaced by the DSR system in GSB. Sequence analyses suggested that the conserved soxJXYZAKBW gene cluster was horizontally acquired by Chlorobium phaeovibrioides DSM 265 from the Chlorobaculum lineage and that this acquisition was mediated by a mobile genetic element. Thus, the last common ancestor of currently known GSB was probably photoautotrophic, hydrogenotrophic, and contained SQR but not DSR or SOX. In addition, the predominance of the Chlorobium-Chlorobaculum-Prosthecochloris lineage among cultured GSB could be due to the horizontally acquired DSR and SOX systems. Finally, based upon structural, biochemical, and phylogenetic analyses, a uniform nomenclature is suggested for sqr genes in prokaryotes.
Prostate cancers show a slow progression from a local lesion (primary tumor) to a metastatic and hormone-resistant phenotype. After an initial step of hyperplasia, in a high percentage of cases a ...neoplastic transformation event occurs that, less frequently, is followed by epithelial to mesenchymal transition and invasion of healthy tissues (usually bones). MicroRNA-203 (miR-203) is a tumor suppressor microRNA often silenced in different malignancies. Here, we show that miR-203 is downregulated in clinical primary prostatic tumors compared to normal prostate tissue, and in metastatic prostate cancer cell lines compared to normal epithelial prostatic cells. Overexpression of miR-203 in brain or bone metastatic prostate cell lines (DU145 and PC3) is sufficient to induce a mesenchymal to epithelial transition with inhibition of cell proliferation, migration and invasiveness. We have identified CKAP2, LASP1, BIRC5, WASF1, ASAP1 and RUNX2 as new miR-203 direct target mRNAs involved in these events. Therefore, miR-203 could be a potentially new prognostic marker and therapeutic target in metastatic prostate cancer.
Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often ...poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3′ end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3′ end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.
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•ELCAP captures RNAPII subcomplexes to facilitate studies of RNAPII-bound factors•Provides single-nucleotide binding profiles and proteomics characterization•Highlights overlapping and separate features of RNAPII-bound SCAF4 and SCAF8
Recently developed approaches allow profiling of RNAPII occupancy at single-nucleotide resolution. However, these methodologies are currently not directly applicable to profile RNAPII subcomplexes co-bound by specific regulatory factors. Here, we have optimized chromatin extraction procedures to preserve interactions between RNAPII and its co-bound factors and used them for a double-affinity purification protocol to capture RNAPII subcomplexes for RNA-seq and proteomics. As we capture hundreds of RNAPII-associated factors in our reference RNAPII proteomics data, we reason that this method can be widely applicable to study RNAPII-associated factors.
Gregersen et al. develop ELCAP, an affinity purification method that enables capture of factor-specific RNA polymerase II elongation complexes for single-nucleotide occupancy profiling and proteomics.
For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria ...(GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline (c. 8 x 10⁶ GSB cells mL⁻¹) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA, and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.
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