scholarly journals Nrd1p identifies aberrant and natural exosomal target messages during the nuclear mRNA surveillance in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Pragyan Singh ◽  
Anusha Chaudhuri ◽  
Mayukh Banerjea ◽  
Neeraja Marathe ◽  
Biswadip Das
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Late Phase ◽  
Vital Role ◽  
Mrna Surveillance ◽  
Normal Functional ◽  
Nuclear Exosome ◽  
Mrnp Biogenesis

AbstractIn all eukaryotes, selective nuclear degradation of aberrant mRNAs by nuclear exosome and its cofactors TRAMP, and CTEXT contribute to the fidelity of the gene expression pipeline. In the model eukaryote, Saccharomyces cerevisiae, the Nrd1p-Nab3p-Sen1p (NNS) complex, previously known to be involved in the transcription termination and matured 3’-end formation of vast majority of non-coding and several coding RNAs, is demonstrated to universally participate in the nuclear decay of various kinds of faulty messages in this study. Consistently, nrd1-1/nrd1-2 mutant cells display impairment of the decay of all kinds of aberrant mRNAs, like the yeast mutants deficient in Rrp41p, Rrp6p, and Rrp4p. nrd1ΔCID mutation (consisting of Nrd1p lacking its CID domain thereby abrogating its interaction with RNAPII) however, abolishes the decay of aberrant messages generated during early phases of mRNP biogenesis (transcription elongation, splicing and 3’-end maturation) without affecting the decay rate of the export-defective mRNAs. Mutation in the 3’-end processing factor, Pcf11p, in contrast, displayed a selective abolition of the decay of the aberrant mRNAs, generated at the late phase of mRNP biogenesis (export-defective mRNAs) without influencing the faulty messages spawned in the early phase of mRNP biogenesis. Co-transcriptional recruitment of Nrd1p onto the faulty messages, which relies on RNAPII during transcription elongation and on Pcf11p post transcription, is vital for the exosomal decay of aberrant mRNAs, as Nrd1p deposition on the export-defective messages led to the Rrp6p recruitment and eventually, their decay. Thus, presence of the ‘Nrd1p mark’ on aberrant mRNAs appears rate-limiting for the distinction of the aberrant messages from their normal functional counterparts.Author’s SummaryAberrant/faulty mRNAs generated from the deficiencies in any of the mRNP biogenesis events are promptly eliminated by the nuclear exosome and its cofactors TRAMP and CTEXT complexes. These machineries work relentlessly in the nucleus to detect all kinds of aberrant mRNAs and selectively target them for destruction. However, initial detection of a minuscule amount of aberrant mRNA in the vast background of normal mRNAs is quite challenging and its mechanism remains elusive. In this work, we demonstrate that, the trimeric Nrd1p-Nab3p-Sen1p complex, previously implicated in the transcription termination of diverse non-coding RNAs and a handful of mRNAs, constitute an integral component of the nuclear mRNA surveillance mechanism in baker’s yeast Saccharomyces cerevisiae. Major component of this complex, Nrd1p is demonstrated to be recruited selectively onto various classes of representative model aberrant messages either co-transcriptionally by RNA Polymerase II or post-transcriptionally by Pcf11p. Binding of Nrd1p to the export-defective special mRNAs further leads to the recruitment of Rrp6p on to them thereby leading to their degradation. NNS complex thus plays a vital role of initially recognizing the faulty messages and further assists in the recruitment of the nuclear exosome for their prompt elimination.

scholarly journals Expression of Bacterial Rho Factor in Yeast Identifies New Factors Involved in the Functional Interplay between Transcription and mRNP Biogenesis

2009 ◽  
Vol 29 (15) ◽  
pp. 4033-4044 ◽  
Author(s):  
Christine Mosrin-Huaman ◽  
Romy Honorine ◽  
A. Rachid Rahmouni
Keyword(s):  
Rna Polymerase Ii ◽  
Growth Defect ◽  
Large Set ◽  
Mrna Synthesis ◽  
Ribonucleoprotein Particle ◽  
Nuclear Exosome ◽  
Dependent Growth ◽  
Dose Dependent ◽  
Dynamic Interplay ◽  
Mrnp Biogenesis

ABSTRACT In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events.

scholarly journals Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein–DNA interactions

1997 ◽  
Vol 69 (2) ◽  
pp. 101-110 ◽  
Author(s):  
ALAN TAYLOR ◽  
LIQUN ZHANG ◽  
JOHN HERRMANN ◽  
BEI WU ◽  
LARRY KEDES ◽  
...  
Keyword(s):  
Protein Binding ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Nuclear Extracts

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to regions 5′ and 3′ of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3′ end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

scholarly journals Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.

1993 ◽  
Vol 13 (2) ◽  
pp. 1296-1305 ◽  
Author(s):  
C A Spencer ◽  
M A Kilvert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Incubation Time ◽  
Transcription Initiation ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Threshold Level ◽  
Specific Effects ◽  
Myc Gene

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc P1 promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase II transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase II processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.

scholarly journals Genomewide Recruitment Analysis of Rpb4, a Subunit of Polymerase II in Saccharomyces cerevisiae, Reveals Its Involvement in Transcription Elongation

2008 ◽  
Vol 7 (6) ◽  
pp. 1009-1018 ◽  
Author(s):  
Jiyoti Verma-Gaur ◽  
Sudha Narayana Rao ◽  
Toshiki Taya ◽  
Parag Sadhale
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Normal Growth ◽  
Growth Conditions ◽  
Pol Ii ◽  
Coding Regions ◽  
The Core ◽  
A Subunit ◽  
On Chip

ABSTRACT The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in Saccharomyces cerevisiae is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in S. cerevisiae and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation (“ChIP on-chip”) technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the rpb4Δ mutant. Unlike most phenotypes of rpb4Δ, the 6AU sensitivity of the rpb4Δ strain was not rescued by overexpression of RPB7. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7.

scholarly journals Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

2020 ◽  
Author(s):  
Victoria Begley ◽  
Antonio Jordán-Pla ◽  
Xenia Peñate ◽  
Ana I. Garrido-Godino ◽  
Drice Challal ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Mrna Decay ◽  
Transcription Elongation ◽  
General Mechanism ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Chromatin Configuration ◽  
Polyadenylation Sites ◽  
Nucleosome Mapping

AbstractmRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5’ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3’ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favoring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.

Intrinsic sites of transcription termination and pausing in the c-myc gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4389-4394
Author(s):  
T K Kerppola ◽  
C M Kane
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
Intron 1 ◽  
Exon 1 ◽  
Myc Gene

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

scholarly journals Saccharomyces cerevisiae Transcription Elongation Mutants Are Defective in PUR5 Induction in Response to Nucleotide Depletion

2000 ◽  
Vol 20 (20) ◽  
pp. 7427-7437 ◽  
Author(s):  
Randal J. Shaw ◽  
Daniel Reines
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Normal Growth ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Pol Ii

ABSTRACT IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective inPUR5 induction. The inability to fully inducePUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5′ untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.

scholarly journals Role of Transcription in Plasmid Maintenance in the hpr1Δ Mutant of Saccharomyces cerevisiae

2002 ◽  
Vol 22 (24) ◽  
pp. 8763-8773 ◽  
Author(s):  
Robert J. Merker ◽  
Hannah L. Klein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Rna Splicing ◽  
Plasmid Stability ◽  
Transcription Elongation ◽  
Transcription Terminator ◽  
Plasmid Instability ◽  
Mature Mrna

ABSTRACT The Saccharomyces cerevisiae hyperrecombination mutation hpr1Δ results in instability of sequences between direct repeats that is dependent on transcription of the repeat. Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation. In the hpr1Δ mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator. The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p. Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1Δ mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA. Suppression of plasmid instability in hpr1Δ can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing. High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA. Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed.

Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes

1993 ◽  
Vol 13 (2) ◽  
pp. 1296-1305
Author(s):  
C A Spencer ◽  
M A Kilvert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Incubation Time ◽  
Transcription Initiation ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Threshold Level ◽  
Specific Effects ◽  
Myc Gene

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc P1 promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase II transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase II processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.

Sign in / Sign up

Export Citation Format

Share Document