DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts.

Precursor messenger RNA (pre-mRNA) is processed into its functional form during RNA polymerase II (Pol II) transcription. Although functional coupling between transcription and pre-mRNA processing is established, the underlying mechanisms are not fully understood. We show that the key transcription termination factor, RNA exonuclease Xrn2 engages with Pol II forming a stable complex.

Studying Exoribonuclease Activity Using Fluorescence Anisotropy Assay.

Fluorescence anisotropy is a powerful technique, widely used for investigating ligand-macromolecule binding and high-throughput screens for drugs. Here, we employ fluorescence anisotropy to quantitatively study the activity of exoribonucleases exemplified by the Xrn2 enzyme. Recording changes in the fluorescence anisotropy over time allows real-time detection of enzymatic activity and provides a framework that can be tailored to particular questions.

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response.

The nuclear RNA exosome plays a key role in controlling the levels of multiple protein-coding and non-coding RNAs. Recruitment of the exosome to specific RNA substrates is mediated by RNA-binding co-factors. The transient interaction between co-factors and the exosome as well as the rapid decay of RNA substrates make identification of exosome co-factors challenging.

Transcription and chromatin-based surveillance mechanism controls suppression of cryptic antisense transcription.

Phosphorylation of the RNA polymerase II C-terminal domain YSPTSPS consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents.

System-wide analyses of the fission yeast poly(A) RNA interactome reveal insights into organization and function of RNA-protein complexes.

Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA-protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A) RNA-protein crosslinking data provide information on the organization of RNA-protein complexes.

Comparative Poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants.

RNA exosome complexes degrade many different RNA substrates. Substrate selection and targeting to the exosome complex rely on cofactors, which bind to the substrate RNA, recruit the exosome complex, and help to remodel the associated ribonucleoprotein particle to facilitate RNA degradation. These cofactors are RNA-binding proteins, but their interaction with RNA may be very transient because the RNAs they are bound to are rapidly turned over by the exosome complex.

Profiling RNA Polymerase II Phosphorylation Genome-Wide in Fission Yeast.

The RNA polymerase II carboxyl-terminal domain (CTD) consists of tandem repeats of consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Dynamic posttranslational modifications of the CTD generate a CTD code crucial for the cotranscriptional recruitment of factors that control transcription, chromatin modification, and RNA processing. Analysis of CTD phosphorylation by ChIP (Chromatin ImmunoPrecipitation) coupled with high-throughput DNA sequencing (ChIP-seq) is a powerful tool to investigate the changes in CTD phosphorylation during the transcription cycle.

Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination.

Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA.

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood.

The regulation and functions of the nuclear RNA exosome complex.

The RNA exosome complex is the most versatile RNA-degradation machine in eukaryotes. The exosome has a central role in several aspects of RNA biogenesis, including RNA maturation and surveillance. Moreover, it is emerging as an important player in regulating the expression levels of specific mRNAs in response to environmental cues and during cell differentiation and development.

Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1.

In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast.

lncRNA recruits RNAi and the exosome to dynamically regulate pho1 expression in response to phosphate levels in fission yeast.

Numerous noncoding transcripts of unknown function have recently been identified. In this study, we report a novel mechanism that relies on transcription of noncoding RNA prt (pho1-repressing transcript) regulating expression of the pho1 gene. A product of this gene, Pho1, is a major secreted phosphatase needed for uptake of extracellular phosphate in fission yeast.

mRNA quality control goes transcriptional.

Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5' capped, spliced and 3' polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC (quality control) mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs.

Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast.

We uncovered a novel role for the spliceosome in regulating mRNA expression levels that involves splicing coupled to RNA decay, which we refer to as spliceosome-mediated decay (SMD). Our transcriptome-wide studies identified numerous transcripts that are not known to have introns but are spliced by the spliceosome at canonical splice sites in Saccharomyces cerevisiae. Products of SMD are primarily degraded by the nuclear RNA surveillance machinery.

The Sm complex is required for the processing of non-coding RNAs by the exosome.

A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3'end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres.

The exosome and heterochromatin : multilevel regulation of gene silencing.

Heterochromatic silencing is important for repressing gene expression, protecting cells against viral invasion, maintaining DNA integrity and for proper chromosome segregation. Recently, it has become apparent that expression of eukaryotic genomesis far more complex than had previously been anticipated. Strikingly, it has emerged that most of the genome is transcribed including intergenic regions and heterochromatin, calling for us to re-address the question of how gene silencing is regulated and re-evaluate the concept ofheterochromatic regions of the genome being transcriptionally inactive.

H3K4 trimethylation by Set1 promotes efficient termination by the Nrd1-Nab3-Sen1 pathway.

In Saccharomyces cerevisiae, the Nrd1-Nab3-Sen1 pathway mediates the termination of snoRNAs and cryptic unstable transcripts (CUTs). Both Nrd1 and the Set1 histone H3K4 methyltransferase complex interact with RNA polymerase II (Pol II) during early elongation, leading us to test whether these two processes are functionally linked. The deletion of SET1 exacerbates the growth rate and termination defects of nrd1 mutants.

The exosome and heterochromatin: multilevel regulation of gene silencing.

Heterochromatic silencing is important for repressing gene expression, protecting cells against viral invasion, maintaining DNA integrity and for proper chromosome segregation. Recently, it has become apparent that expression of eukaryotic genomes is far more complex than had previously been anticipated. Strikingly, it has emerged that most of the genome is transcribed including intergenic regions and heterochromatin, calling for us to re-address the question of how gene silencing is regulated and re-evaluate the concept of heterochromatic regions of the genome being transcriptionally inactive.

Synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxyandrost-5-en-17-one via microbial 7alpha-hydroxylation.

The synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7alpha-hydroxylation has been accomplished. At the first stage, 3beta,7alpha-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion.

The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain.

RNA polymerase II (Pol II) in Saccharomyces cerevisiae can terminate transcription via several pathways. To study how a mechanism is chosen, we analyzed recruitment of Nrd1, which cooperates with Nab3 and Sen1 to terminate small nucleolar RNAs and other short RNAs. Budding yeast contains three C-terminal domain (CTD) interaction domain (CID) proteins, which bind the CTD of the Pol II largest subunit.

Transcription termination and RNA degradation contribute to silencing of RNA polymerase II transcription within heterochromatin.

Within the heterochromatin of budding yeast, RNA polymerase II (RNAPII) transcription is repressed by the Sir2 deacetylase. Although heterochromatic silencing is generally thought to be due to limited accessibility of the underlying DNA, there are several reports of RNAPII and basal transcription factors within silenced regions. Analysis of the rDNA array revealed cryptic RNAPII transcription within the "nontranscribed" spacer region.

Distinct pathways for snoRNA and mRNA termination.

Transcription termination at mRNA genes is linked to polyadenylation. Cleavage at the poly(A) site generates an entry point for the Rat1/Xrn2 exonuclease, which degrades the downstream transcript to promote termination. Small nucleolar RNAs (snoRNAs) are also transcribed by RNA polymerase II but are not polyadenylated.

Nrd1 interacts with the nuclear exosome for 3' processing of RNA polymerase II transcripts.

The exosome complex is involved in multiple RNA processing and degradation pathways. How exosome is recruited to particular RNA substrates and then chooses between RNA processing and degradation modes remains unclear. We find that the RNA binding protein Nrd1, complexed with its partners Nab3, Sen1, and cap binding complex, physically interacts with the nuclear form of exosome.

The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNApII) largest subunit consists of multiple heptapeptide repeats with the consensus sequence YSPTSPS. Different CTD phosphorylation patterns act as recognition sites for the binding of various messenger RNA processing factors, thereby coupling transcription and mRNA processing. Polyadenylation factors are co-transcriptionally recruited by phosphorylation of CTD serine 2 (ref.