Subunit Of RNA Polymerase II Research Articles

What causes flag smut of wheat?

The causal agent of flag smut of wheat is currently subject to strict quarantine regulations in many countries and is believed to have a wide host range on wild and cultivated grasses. This fungus has been classified as both Urocystis agropyri and Urocystis tritici. Urocystis agropyri was first described from Elymus repens in Germany and U. tritici was first described from Triticum vulgare (=T. aestivum). In 1953, G. W. Fischer placed U. tritici and a large number of other Urocystis species in synonymy with U. agropyri. The present study is the first attempt to clarify the taxonomy and phylogeny of flag smut pathogens of grasses using molecular analyses. Three loci, the internal transcribed spacer (ITS) region of rDNA, the RNA polymerase II subunit 2 (RPB2), and translation elongation factor (TEF) protein‐coding regions were used for phylogenetic reconstruction to determine the species boundaries of 24 Urocystis specimens from triticoid hosts. Results indicate that there are several distinct lineages of flag smut pathogens, including the causal agent of flag smut of wheat, which is supported as a separate species, U. tritici. Sequences from specimens on E. repens, which are retained as U. agropyri, grouped in a clade distinct from those on wheat and rye. The closest relatives of U. tritici were found to be U. hispanica from Aegilops and Urocystis sp. from Thinopyrum junceiforme and Elymus trachycaulis. Recognition that U. tritici is genetically distinct from U. agropyri sensu stricto will impact regulatory policy and facilitate the development of diagnostic tests.

Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA.

Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells.

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications.

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown. We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs. Our data demonstrated that RPAP2, and to a lesser extent GPN1/RPAP4, are accumulated focally in the cytoplasm of MFM muscle fibres in which they co-localize with POLR2A/RPB1, the largest subunit of RNAPII, and correspond to αB-cystallin deposits in distribution and staining intensity. No abnormal staining for RPAP2 has been observed in muscle of patients with central cores, minicores and neurogenic target fibres. Together, these findings could provide new insights into the molecular pathogenesis of MFMs and suggest that RPAP2 immunostaining can be a useful diagnostic tool to depict protein aggregates in MFMs.

Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.

Selective Dephosphorylation by SCP1 and PP2A in Phosphorylated Residues of SMAD2

E-mail: ykim@kku.ac.krReceived July 1, 2014, Accepted July 31, 2014Key Words : Small CTD phosphatases, SCP1, SMAD2, Dephosphorylation, SMAD phosphataseProtein phosphatase is important for cellular events, and afamily of protein phosphatases, the so-called C-terminaldomain (CTD) of RNA polymerase II (RNAPII) phos-phatases, has recently attracted attention. The CTD is thelargest subunit of RNAPII and consists of a tandem repeatedheptapeptide (Y

Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1.

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that—consistent with previous data—DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII.

The C-Terminal Domain of Rpb1 Functions on Other RNA Polymerase II Subunits

The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.

Mutual relationships between transcription and pre‐mRNA processing in the synthesis of mRNA

The generation of messenger RNA (mRNA) in eukaryotes is achieved by transcription from the DNA template and pre-mRNA processing reactions of capping, splicing, and polyadenylation. Although RNA polymerase II (RNAPII) catalyzes the synthesis of pre-mRNA, it also serves as a principal coordinator of the processing reactions in the course of transcription. In this review, we focus on the interplay between transcription and cotranscriptional pre-mRNA maturation events, mediated by the recruitment of RNA processing factors to differentially phosphorylated C-terminal domain of Rbp1, the largest subunit of RNAPII. Furthermore, we highlight the bidirectional nature of the interplay by discussing the impact of RNAPII kinetics on pre-mRNA processing as well as how the processing events reach back to different phases of gene transcription.

Endothelin-1­induced cardiomyocyte hypertrophy is partly regulated by transcription factor II-F interacting C-terminal domain phosphatase of RNA polymerase II

AimsCardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance with RNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit of RNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic response in cardiomyocytes. However, it is unclear whether ET-1-induced hypertrophy is affected by the CTD phosphatase, transcription factor IIF-interacting CTD phosphatase1 (FCP1). Main methodsWe analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpression of FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes. Key findingsThe level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression, whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [3H]-uridine incorporation showed that the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, the ET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, was suppressed by FCP1 and was not inhibited by dnFCP1. SignificanceET-1-induced cardiac hypertrophy and CTD phosphorylation level are functionally regulated by FCP1. These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controlling phosphorylation level of the RNAPII CTD.

The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organisms.

The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

BackgroundCellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.ResultsWe first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.ConclusionsWe demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

Human GTPases Associate with RNA Polymerase II To Mediate Its Nuclear Import

Small GTPases share a biochemical mechanism and act as binary molecular switches. One important function of small GTPases in the cell is nucleocytoplasmic transport of both proteins and RNA. Here, we show the stable association of human GPN1 and GPN3, small GTPases related to Ran, with RNA polymerase II (RNAPII) isolated from either the cytoplasmic or nuclear fraction. GPN1 and GPN3 directly interact with RNAPII subunit 7 (RPB7)/RPB4 and the C-terminal domain (CTD) of RNAPII. Depletion of GPN1 or GPN3 using small interfering RNAs led to decreased RNAPII levels in the nucleus and an accumulation of this enzyme in the cytoplasm of human cells. Furthermore, isolation of a GPN1/GPN3/RNAPII complex from stable cell lines expressing a dominant negative GPN1 harboring mutations in the GTP-binding pocket demonstrated a role for these proteins in nuclear import of RNAPII. Thus, GPN1/GPN3 define a new family of small GTPases that are specialized for the transport of RNA polymerase II into the nucleus.

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2–Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription.

MOLECULAR EVIDENCE OF TAPHRINA WIESNERI IN LEAVES AND BUDS OF HEALTHY SWEET CHERRY: A POSSIBLE ENDOPHYTISM?

Analysis of DNAs from leaves and buds of several sweet cherry trees (Prunus avium) in two different Italian regions with different climates, has revealed the presence of Taphrina wiesneri, in all samples analyzed. The presence of other fungi was only occasional. The presence of T. wiesneri was shown by sequencing segments of rDNA genes amplified by PCR. Analysis of genes coding for the elongation factor-1a (EF1-a) and RNA-polymerase II subunit 1 (RPB1), confirmed the result. The T. wiesneri sequences were variants of those reported in data bases. T. wiesneri was not detected in buds of walnut, apricot and sour cherry growing close to the analysed sweet cherry trees. Determination of the relative numbers of T. wiesneri and sweet cherry genomes in samples prepared from the same buds of 4 different trees all gave similar values indicating massive presence of the fungus. However, in situ hybridization experiments on bud sections using a T. wiesneri-specific 18S rDNA probe and stains for fungi, labelled structures not clearly representing hyphae.

Mediator Structure and Interaction with the Basal Transcription Machinery

Understanding the structure and conformational dynamics of Mediator is critical for unraveling the essential role the complex plays in regulation of eukaryotic transcription initiation. We used single particle macromolecular electron microscopy (EM) to study the conformational variability of Mediator and that information enabled us to determine a cryo‐EM structure of the complex at ~26Å resolution. Comparing the structures of free and RNA polymerase II (RNAPII)‐associated Mediator made possible a detailed description of Mediator structural modules and the rearrangement they undergo upon interaction with RNAPII, both of which appear to be conserved from yeast to humans. We have also pursued independent EM analysis of the Head module responsible for control of the Mediator‐RNAPII interaction. Conformational analysis of a recombinant Head module revealed distinct conformations and comparison with the structures of sub‐complexes provided information about subunit organization. All of this information, along with results from biochemical and structural analysis of the interaction of the Head module with the TATA‐binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7, suggest a possible mechanism for Mediator regulation of RNAPII interaction with promoter DNA.Supporteb by NIH R01 GM67167

Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1-Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.