FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1921052768> ?p ?o ?g. }

• W1921052768 endingPage "1964" @default.
• W1921052768 startingPage "1953" @default.
• W1921052768 abstract "Article8 April 2011free access A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors Cristina Tous Cristina Tous Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Ana G Rondón Ana G Rondón Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author María García-Rubio María García-Rubio Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Cristina González-Aguilera Cristina González-Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Rosa Luna Rosa Luna Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Andrés Aguilera Corresponding Author Andrés Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Cristina Tous Cristina Tous Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Ana G Rondón Ana G Rondón Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author María García-Rubio María García-Rubio Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Cristina González-Aguilera Cristina González-Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Rosa Luna Rosa Luna Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Andrés Aguilera Corresponding Author Andrés Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain Search for more papers by this author Author Information Cristina Tous1,‡, Ana G Rondón1,‡, María García-Rubio1, Cristina González-Aguilera1, Rosa Luna1 and Andrés Aguilera 1 1Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain ‡These authors contributed equally to this work *Corresponding author. Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Américo Vespucio s/n, P.C.T. Cartuja 93, Sevilla 41092, Spain. Tel.: +34 954 468372; Fax: +34 954 461664; E-mail: [email protected] The EMBO Journal (2011)30:1953-1964https://doi.org/10.1038/emboj.2011.109 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation. Introduction Eukaryotic RNA polymerase II (RNAPII)-mediated transcription elongation is a dynamic and highly regulated stage of the transcription cycle that is functionally linked to mRNA processing, including capping, splicing, 3′-end processing, surveillance and export. The carboxy-terminal domain (CTD) of the RNAPII has a key role in this coupling, which is necessary for the efficient formation of an export competent ribonucleoparticle (mRNP) (Bentley, 2005; Luna et al, 2008; Selth et al, 2010). During elongation, specific factors associate with RNAPII modulating its catalytic activity to help it overcome situations derived from transient pausing, arrest and termination. In addition, elongation through chromatin demands nucleosome remodelling and/or histone modification via functionally specialized factors (Li et al, 2007). In the last few years, a number of factors acting at different levels in mRNP biogenesis and export have been reported to impact on transcription elongation. However, whether or not many of these factors have an active role in elongation in vivo is yet elusive. Genetic and biochemical approaches, both in vivo and in vitro, have been used to study transcription elongation. In vitro there are at least two types of methods for the analysis of transcription elongation. The first is based on purified RNA polymerase engaging elongation directly on an oligonucleotide with a dC-tail (Kadesch and Chamberlin, 1982). The second one was set for the analysis of elongation in naked DNA using yeast whole cell extracts (WCEs) and a plasmid with two G-less cassettes (Rondon et al, 2003). Different in vivo methods have been used to study transcription elongation. In transcriptional run-on assays, the nascent pre-mRNA is labelled with a pulse of radioactive UTP in permeabilized cells. RNA is then analysed by hybridization to immobilized strand-specific probes (Warner, 1991). Another method extensively employed is chromatin immunoprecipitation (ChIP) analyses of RNAPII distribution across a gene (Mason and Struhl, 2005). None of these assays consider that elongation might be differently affected depending on the sequence, the GC:AT content or the length of the DNA template, even though these features have been shown to influence the efficiency of transcription elongation (Chavez et al, 2000; Gallardo and Aguilera, 2001; Rondon et al, 2003, 2004). Conversely, although constructs based on open reading frames (ORFs) with different length and GC content placed under a GAL1 promoter have been used to infer elongation efficiency by northern analysis (Chavez et al, 2001; Luna et al, 2005), they only provide a first but not definitive answer as the results do not exclude a putative impact of RNA stability. Despite the availability of different in vivo assays, their lack of specificity on transcription elongation yield results that in some cases are unclear or different depending on the assay employed. That is the case of the PAF and THSC complexes. PAF is a five-subunit complex containing Paf1, Cdc73, Ctr9, Rtf1 and Leo1, which seems to orchestrate different mRNP biogenesis processes. It coordinates chromatin modification during transcription elongation via interaction with histone methylases and ubiquitinylases (Krogan et al, 2003; Wood et al, 2003), and it is involved in the 3′-end formation of polyadenylated and non-polyadenylated RNAPII transcripts (Penheiter et al, 2005; Sheldon et al, 2005). The role of PAF in transcription elongation was proposed on the basis of its physical interaction with RNAPII, genetic and physical interactions with factors such as Spt4–Spt5 and Spt16–Pob3, and its recruitment to the ORF of transcribed genes (Costa and Arndt, 2000; Krogan et al, 2002; Pokholok et al, 2002; Squazzo et al, 2002). Mutations in the Paf1 and Cdc73 subunits, but not in Rtf1 and Leo1, reduce the transcription-elongation efficiency in vitro (Rondon et al, 2004). However, loss of PAF components does not result in an altered distribution of elongating RNAPII (Mueller et al, 2004; Mason and Struhl, 2005). THSC, also termed TREX-2, is a conserved multifunctional complex formed by Thp1, Sac3, Sus1 and Cdc31, which works at the transcription–mRNA export interface as defined in the yeast Saccharomyces cerevisiae. THSC is located at the nuclear pore complex (NPC) and is required for mRNA export and gene tethering (Fischer et al, 2002, 2004; Gallardo et al, 2003; Rodriguez-Navarro et al, 2004; Cabal et al, 2006). Northern and ChIP analyses of long and GC-rich sequences in THSC mutants point to a role in transcription elongation. However, this has not been corroborated by the G-less in vitro assay (Gonzalez-Aguilera et al, 2008). As THSC is located at the nuclear periphery, it is an open question whether the effect of this complex on transcription is only relevant when coupled to mRNA export, but not in cell extracts in which the nuclear membrane is disrupted. Other factors with known functions in transcription initiation or mRNA metabolism recently shown to be involved in elongation are several subunits of the SAGA complex, the Rrp6 component of the nuclear exosome, and subunits of the mRNA 3′-end processing complex cleavage factor IA (CFIA) (Luna et al, 2005; Govind et al, 2007). In order to clarify and ascertain the role of these factors in transcription elongation, we developed an in vivo assay for a direct and sensitive analysis of transcription elongation on chromatin and in an intact nuclear structure. Importantly, this assay takes in consideration different features of the template that influence elongation as the length and GC content. After validating the assay with known bona fide transcription-elongation mutants such as spt4 and rpb9, we analysed a number of mutants of the PAF, THSC and SAGA complexes, the nuclear exosome, and the 3′-end processing complex CFIA. Taking advantage of this assay, we undertook a genetic screen for putative transcription-elongation mutants and showed that the Nup84 complex of the NPC is required for transcription elongation. Our results, in addition to showing that the G-less-based run-on assay (GLRO) assay is a highly reliable method for the in vivo analysis of transcription elongation, provide novel and unambiguous conclusions about the involvement of the analysed factors in transcription elongation and serves to define a new role for the Nup84 complex in RNAPII elongation. This strengthens the idea of a functional relationship between nuclear export and transcription elongation, demonstrating an impact of the NPC in transcription elongation. Results A new G-less cassette-based run-on assay, GLRO, for the direct analysis of transcription elongation in vivo We have developed a run-on assay for transcription elongation that allows quantification of nascent mRNA directly, without any need of hybridization or PCR amplification. The assay is based on a set of two constructs that differ in the length and GC content of the DNA sequence to be transcribed. The comparative analysis of the transcription-elongation efficiency through a long versus a short DNA fragment is essential as long transcripts are more sensitive to defects in elongation than short ones. We will refer to this assay as GLRO (G-less cassette-based run-on assay). The GLRO constructs are based on the CYCds plasmid (Steinmetz and Brow, 2003) bearing two G-less cassettes of 262 nt and 132 nt separated by a 243-nt CYC1 fragment as spacer sequence. This construct is transcribed from the strong constitutive TDH3 promoter. In this plasmid, we cloned a 2-kb fragment of the LacZ gene between the two G-less cassettes to generate the CYC-LacZ construct. The length and high GC content of lacZ makes transcription through this sequence poorly efficient in mutants impairing elongation (Chavez et al, 2000; Rondon et al, 2003). The GLRO assay was first performed in wild-type cells transformed with the plasmid-borne CYCds and CYC-lacZ constructs, from now on termed GLRO-short and GLRO-long, respectively (Figure 1A). Briefly, after in vivo labelling of the nascent mRNA in the run-on reaction, the resulting transcripts were purified and treated with RNase T1 to degrade all G-containing sequences, rendering the two G-less cassettes as two intact fragments that were resolved by polyacrylamide gel electrophoresis (PAGE). Transcription-elongation efficiency was measured as the ratio of 32P incorporated into the 132-nt-long versus the 262-nt-long G-less cassette for each construct. We confirmed by northern that the lacZ inserted between the two G-less cassettes in GLRO-long is correctly transcribed in wild-type cells (data not shown). GLRO values in the different mutants are normalized to the corresponding wild-type levels for both the GLRO-short and GLRO-long constructs. Figure 1.Characterization of the G-less cassette-based run-on transcription-elongation assay (GLRO) in wild-type and spt4Δ and rpb9Δ mutants. (A) Scheme of the tandem G-less cassette constructs used for GLRO analysis. Black rectangles represent sequences derived from CYC 3′-flanking region, and the white and grey rectangles correspond to the two G-less cassettes and the 2-kb lacZ fragment, respectively. (B) GLRO analysis of wild-type and spt4Δ and rpbΔ9 mutants transformed with the GLRO-short and GLRO-long systems. Transformants were grown in SC-leu medium to exponential phase and run-on transcription assays were performed as described in Materials and methods. The transcription run-on products were digested with RNase T1 and resolved in a 6% PAGE. A representative acrylamide gel is shown. For each sample, the ratio of total counts incorporated into the distal versus the proximal G-less cassette was normalized against the ratio for the same construct in the wild-type strain. The mean value and s.d. of three independent experiments are shown. Download figure Download PowerPoint To validate the GLRO assay, we measured transcription elongation in null mutants of genes with a known role in transcription elongation like SPT4 and RPB9. Spt4 (Spt4–Spt5/DSIF complex) is involved in RNAPII promoter-proximal pausing and it also has a positive role in transcription elongation as shown by 6-azauracil (6-AU) sensitivity, northern analysis, RNAPII ChIP and in vitro transcription-elongation assays (see Supplementary Table I; Hartzog et al, 1998; Rondon et al, 2003). Rpb9 is a non-essential subunit of RNAPII that functions in transcription initiation and elongation and is important for transcription fidelity (Hull et al, 1995; Hemming et al, 2000; Nesser et al, 2006). Rpb9 in vivo relevance in elongation was determined by 6-AU sensitivity and by its ability to interact with the transcription-elongation factor TFIIS (Awrey et al, 1997; Hemming et al, 2000). Transcription-elongation efficiency (transcription of the second G-less cassette versus the first one) of spt4Δ was 30% of the wild type for the GLRO-long and similar to the wild type for the GLRO-short construct (Figure 1B). This result is consistent with the decrease in efficiency (<25% with respect to the wild type) previously determined in vitro with an assay based on other two G-less cassette constructs (Rondon et al, 2003). Next, we assayed the transcription-elongation efficiency of rpb9Δ, using both the in vivo run-on systems (Figure 1B) and the in vitro G-less assay (Supplementary Figure S1). In this case, the rpb9Δ mutant reduces transcription elongation to 60–70% of the wild-type values in both the GLRO-long and GLRO-short systems. In addition, a reduction to 50% of the wild-type elongation rate was also observed in vitro using rpb9Δ WCEs (Supplementary Figure S1). Together, these results indicate that the GLRO assay is sensitive enough to detect a decrease in elongation rate. Transcription elongation is defective in PAF mutants The PAF complex has several roles in mRNP biogenesis from transcription elongation to transcription termination and 3′-end mRNA processing (Mueller et al, 2004; Penheiter et al, 2005; Sheldon et al, 2005). Evidence supports a hierarchical and specialized role for the different subunits. It has previously been shown that ablation of any of the PAF-subunit genes severely decreased the mRNA levels of the long and GC-rich lacZ ORF driven from a GAL1 promoter, whereas it did not affect transcription of short mRNAs driven from that same promoter (Rondon et al, 2004). These data suggest a defect in transcription elongation rather than in initiation, otherwise the defect should have been the same for both constructs. However, analysis of in vitro transcription elongation indicated that only two subunits, Paf1 and Cdc73, had a detectable function in transcription elongation (Rondon et al, 2004). In contrast, when transcription elongation was assessed in vivo by RNAPII distribution across a long gene, no defect was observed in any of the PAF mutants (Mason and Struhl, 2005). Therefore, we decided to test the effect of different PAF mutants in the GLRO assays. The paf1Δ, cdc73Δ and rtf1Δ strains showed a slight reduction in the GLRO-short system and a stronger reduction in the GLRO-long with levels of 50–60% of the wild type (Figure 2). Transcription levels were also slightly reduced the GLRO-long system in leo1Δ, although not as strongly as in the other mutants. Overall, our results confirm that, indeed, PAF has a clear role in transcription elongation in vivo. Figure 2.GLRO analysis of PAF mutants. Wild-type (BY4741), paf1Δ, rtf1Δ, cdc73Δ and leo1Δ isogenic strains were transformed with the GLRO-short and GLRO-long constructs and transcription run-on assays were performed. A representative assay is shown. The mean value and s.d. of three independent experiments are shown. Other details are as in Figure 1. Download figure Download PowerPoint Differential effect of THSC mutations in transcription elongation THSC mutants are impaired in transcription elongation in vivo, as determined by northern analysis of long versus short genes and RNAPII ChIPs, but they fail to show a significant elongation defect in vitro in contrast to other mutants with similar in vivo effects (Gallardo et al, 2003; Gonzalez-Aguilera et al, 2008). Therefore, we sought to analyse transcription in THSC mutants with the GLRO assay. Interestingly, the sac3Δ and thp1Δ strains diminished transcription-elongation efficiency to 50–60% of the wild-type levels only in the GLRO-long construct, whereas sus1Δ showed no decrease (Figure 3A). These results confirm that THSC has a role in transcription elongation that is only appreciated in vivo when transcription and mRNA export are coupled. Figure 3.Analysis of transcription elongation in THSC. (A) GLRO analysis of THSC mutants. Transcription run-on assays of thp1Δ, sac3Δ, sus1Δ, mutants carrying the plasmids GLRO-short and GLRO-long. (B) RNAPII occupancy in sus1Δ mutant. ChIP analyses in wild-type (BY4741), thp1Δ and sus1Δ strains transformed with the pLAUR expression system. ChIP analyses in wild-type, thp1Δ and sus1Δ strains carrying the GAL1p::YLR454w fusion construct located at the endogenous YLR454w chromosomal locus. The scheme of the gene and the PCR-amplified fragments are shown. Numbers indicate the primer position respect to the first ATG of the gene. The sequence is provided in Supplementary Table II. The DNA ratios between the 5′ and 3′ regions were calculated from their signal relative to the signal of the intergenic region. The recruitment data shown refer to the value of the 5′ region normalized to 100%. ChIPs were performed from three independent cultures, and quantitative PCRs were repeated three times for each culture. Download figure Download PowerPoint Sus1 is a factor present in two protein complexes: THSC and SAGA, a complex involved in transcription initiation of a subset of genes (Rodriguez-Navarro et al, 2004). As previous studies suggested that sus1Δ has a defect in transcription elongation in vivo (Pascual-Garcia et al, 2008), we wanted to confirm by other means the lack of effect shown by sus1Δ in the GLRO assay. Thus, we measured RNAPII occupancy across the pLAUR expression system that contains a 4.15-kb lacZ-URA3 translational fusion under the tet promoter (Figure 3B) (Jimeno et al, 2002). THSC mutants transcribe poorly the pLAUR construct, as shown by northern (Gallardo et al, 2003; Gonzalez-Aguilera et al, 2008). Indeed, RNAPII occupancy at the 3′-end of lacZ gene was reduced 50% with respect to the 5′-end in thp1Δ, consistent with previous in vivo data. Importantly, no reduction was observed in a sus1Δ mutant. Similarly, ChIP analyses in the YLR454w gene fused to the GAL1 promoter showed a decrease in the RNAPII signal at the 3′-end in thp1Δ cells but not in sus1Δ cells (Figure 3B). These results differ from the small decrease in RNAPII level reported for the YLR454w ORF, probably due to experimental conditions (Pascual-Garcia et al, 2008). We conclude that Sus1, even though it is recruited along the ORF of genes in a transcription-dependent manner (Pascual-Garcia et al, 2008), does not significantly affect transcription elongation. SAGA mutations that impair transcription elongation SAGA is a well-known transcription initiation factor with a possible role in elongation as several of its components are recruited along the genes (Govind et al, 2005, 2007). In order to explore this possibility, we decided to extend our analyses to some SAGA mutants, such as gcn5Δ, spt20Δ and sgf73Δ (Figure 4A). Gcn5 is an acetyltransferase, Spt20 has a role in the structural integrity of SAGA and Sgf73 is a subunit of the histone-deubiquitinating module that interacts with THSC (Candau et al, 1997; Grant et al, 1997; Kohler et al, 2008). Mutations in these factors did not substantially affect GLRO-short transcription with the exception of spt20Δ. However, in the GLRO-long construct we appreciated a decrease of the elongation rate in the three mutants (Figure 4A). These data are in agreement with the reduced RNAPII occupancy at the 3′-end of specific genes in gcn5 and spt20 mutants (Govind et al, 2005; Gaillard et al, 2009), and with the low efficiency of spt20Δ to transcribe in vitro the two G-less cassette system (<60% with respect to the wild type) (Gaillard et al, 2009). In summary, these results support the view of SAGA as a histone-modification complex implicated in transcription initiation and also in transcription elongation. It remains to be seen whether these subunits influence elongation as part of SAGA or independently. Figure 4.GLRO analysis of mutants of the SAGA, nuclear exosome and 3′-end processing factors. (A) GLRO analysis of SAGA mutants. Transcription run-on assays of wild-type, sgf73Δ, gcn5Δ and spt20Δ strains carrying GLRO-short and GLRO-long plasmids are shown. (B) GLRO analysis of nuclear exosome and 3′-end processing mutants. rrp6Δ, rna14-1, rna15-1, and a isogenic wild-type strain were transformed with the GLRO-short and GLRO-long plasmids and transcription run-on assays were performed. A representative assay is shown. The mean value and s.d. of three independent experiments are shown. Other details are as in Figure 1. Download figure Download PowerPoint Transcription elongation diminishes in mutants of nuclear exosome components or 3′-end processing factors Finally, we determined with the GLRO assay the transcription-elongation efficiency of mutants with previously shown in vivo and in vitro elongation defects, which were particularly intriguing given their known role in different mRNA metabolism steps. In particular, we analysed the implication of 3′-end mRNA processing and termination factors Rna14 and Rna15 and the nuclear exosome component Rrp6. In these three mutants, the transcription-elongation rate was notably diminished to 40–50% of the wild-type level in the GLRO-long assay, whereas no difference was observed in the GLRO-short assay (Figure 4B). These results are consistent with the decrease in the RNAPII occupancy observed by ChIP analysis and with the defect on transcription elongation reported in vitro with the two G-less assay (Luna et al, 2005). Therefore, the GLRO assays unambiguously identify CFIA and the nuclear exosome component Rrp6 as two factors that confer high efficiency of transcription elongation in vivo. The fact that the effect is only observed in the GLRO-long system suggests that the activity of these factors is only relevant in a late elongation stage as it seems to be the case also for Spt4 and THSC. A novel role for the Nup84 complex in transcription elongation As mentioned above, transcription through the bacterial lacZ gene in yeast is especially sensitive to mutations in elongation factors. Thus, mutations in several of the complexes required for elongation, as stated by the GLRO assay, also diminished lacZ mRNA accumulation in northern assays (Luna et al, 2005). Having developed the new GLRO assay as a sensitive method to assess the in vivo transcription-elongation efficiency, we sought to perform a genetic screen to identify new factors required for transcription elongation. By employing a Tn3 deletion library (Burns et al, 1994), we mutated a strain with a GAL1p::LacZ fusion integrated in Chromosome V and searched for a decrease in β-galactosidase (β-gal) activity. In all, 6160 colonies were screened by β-gal colony colour assay. After several selection steps that included quantification of β-gal activity, only three mutants unambiguously reduced the β-gal activity to 40% the wild-type level. However, in only one of the mutants, the reduction of β-gal co-segregated with the Tn3 marker and, therefore, was genetically linked to the Tn3 insertion. This low yield of candidates may be due to both a low genomic representation in the library used and, above all, the poor sensitivity of the β-gal colony colour assay, in contrast to northern analysis, to detect a decrease in lacZ transcription. Cloning and sequencing of the candidate revealed that the Tn3 transposon was inserted at the NUP84 promoter, disrupting its activity. Therefore, we continued the analysis with a nup84Δ strain. For further genetic evidence of the Nup84 involvement in RNAPII elongation in vivo we searched for possible genetic interactions of Nup84 with other factors implicated in elongation. As can be seen in Figure 5A, the double mutants nup84Δ dst1Δ and nup84Δ spt4Δ are viable in contrast to the double mutants nup84Δ sac3Δ consistent with non-overlapping roles of the Nup84 and THSC complexes in mRNA export. However, nup84Δ dst1Δ and nup84Δ spt4Δ increased the sensitivity to mycophenolic acid (MPA), a drug that compromises transcription elongation, with respect to the single mutants (Figure 5B). These results are, therefore, consistent with a role of Nup84 in RNAPII transcription elongation in vivo. Figure 5.Genetic interactions of Nup84 with transcription-elongation and mRNA export factors. (A) Tetrad analyses of different crosses between nup84Δ and dst1Δ, spt4Δ and sac3Δ strains. Circles indicate the positions of the double mutants. Only the double nup84Δ sac3Δ are inviable. (B) Micophenolic acid (MPA) sensitivity of nup84Δ, dst1Δ and spt4Δ single and double mutants as determined by 10-fold serial dilutions on SC plates containing the indicated concentrations of MPA. Download figure Download PowerPoint To obtain molecular evidence of the Nup84 effect in transcription elongation, we first confirmed by northern analysis that lacZ transcription was reduced in a nup84Δ mutant (data not shown). Then, we determined by ChIP the RNAPII levels at the pLAUR system. As expected, the observed decrease in lacZ mRNA correlates with a reduction in RNAPII occupancy at the 3′-end of the pLAUR system versus the 5′-end (Figure 6A), indicating that Nup84 contributes to RNAPII transcription elongation through the bacterial lacZ gene. Similar results were obtained in the endogenous HSP104 gene and the GLRO-long system, indicating that the effect in elongation is not sequence specific (Figure 6B and C). The reduction of RNAPII occupancy observed at the 3′-end region of HSP104 in the nup84Δ mutant was as severe as in the spt4Δ or thp1Δ (50%) while in the GLRO-long system it was less pronounced (70%) (Figure 6B and C). Altogether the RNAPII distribution analysis is consistent with a general defect in transcription elongation. Figure 6.RNAPII distribution along different genes in nucleoporin mutants. (A) RNAPII ChIP analysis in the pLAUR system in the nup84Δ and an isogenic wild-type strains. (B) RNAPII ChIP analysis in the HSP104 endogenous gene in the nup84Δ, thp1Δ and spt4Δ mutants and their isogenic wild-type strain (C) RNAPII ChIP analysis in the GLRO-long system in the nup84Δ, thp1Δ and spt4Δ mutants and their isogenic wild-type strain. Other details are as in Figure 3. Download figure Download PowerPoint Next, we took advantage of the newly developed GLRO assay to directly determine the impact of nup84Δ in transcription elongation. As can be seen in Figure 7A, nup84Δ deletion affects elongation through the GLRO-long construct although to a lesser extent than mutations in PAF, THSC or Spt4 (70% the wild-type level in nup84Δ and 30–50% in PAF, THSC or Spt4 mutants). These results ar" @default.
• W1921052768 created "2016-06-24" @default.
• W1921052768 creator A5030807192 @default.
• W1921052768 creator A5043281613 @default.
• W1921052768 creator A5054797076 @default.
• W1921052768 creator A5060936801 @default.
• W1921052768 creator A5063444330 @default.
• W1921052768 creator A5069444674 @default.
• W1921052768 date "2011-04-08" @default.
• W1921052768 modified "2023-10-02" @default.
• W1921052768 title "A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors" @default.
• W1921052768 cites W1548336866 @default.
• W1921052768 cites W1568843108 @default.
• W1921052768 cites W1848777874 @default.
• W1921052768 cites W1943761658 @default.
• W1921052768 cites W1966017154 @default.
• W1921052768 cites W1968789240 @default.
• W1921052768 cites W1972029047 @default.
• W1921052768 cites W1983848119 @default.
• W1921052768 cites W1987911801 @default.
• W1921052768 cites W1992016490 @default.
• W1921052768 cites W1996183355 @default.
• W1921052768 cites W2002085309 @default.
• W1921052768 cites W2004828705 @default.
• W1921052768 cites W2014105270 @default.
• W1921052768 cites W2015298553 @default.
• W1921052768 cites W2019379779 @default.
• W1921052768 cites W2019923915 @default.
• W1921052768 cites W2027592091 @default.
• W1921052768 cites W2030197516 @default.
• W1921052768 cites W2033244618 @default.
• W1921052768 cites W2035946637 @default.
• W1921052768 cites W2036947100 @default.
• W1921052768 cites W2038711435 @default.
• W1921052768 cites W2044313365 @default.
• W1921052768 cites W2051333710 @default.
• W1921052768 cites W2052586663 @default.
• W1921052768 cites W2053416801 @default.
• W1921052768 cites W2054555589 @default.
• W1921052768 cites W2061913428 @default.
• W1921052768 cites W2062630554 @default.
• W1921052768 cites W2069200751 @default.
• W1921052768 cites W2071837083 @default.
• W1921052768 cites W2079493448 @default.
• W1921052768 cites W2079815614 @default.
• W1921052768 cites W2081022101 @default.
• W1921052768 cites W2081995991 @default.
• W1921052768 cites W2084056048 @default.
• W1921052768 cites W2084389717 @default.
• W1921052768 cites W2086086978 @default.
• W1921052768 cites W2091997786 @default.
• W1921052768 cites W2097438415 @default.
• W1921052768 cites W2098184015 @default.
• W1921052768 cites W2100041522 @default.
• W1921052768 cites W2100132835 @default.
• W1921052768 cites W2100313737 @default.
• W1921052768 cites W2101461473 @default.
• W1921052768 cites W2105721989 @default.
• W1921052768 cites W2107635128 @default.
• W1921052768 cites W2109052166 @default.
• W1921052768 cites W2109054128 @default.
• W1921052768 cites W2109845157 @default.
• W1921052768 cites W2110524124 @default.
• W1921052768 cites W2115125406 @default.
• W1921052768 cites W2119470870 @default.
• W1921052768 cites W2122000770 @default.
• W1921052768 cites W2124624490 @default.
• W1921052768 cites W2126422132 @default.
• W1921052768 cites W2132760358 @default.
• W1921052768 cites W2133452708 @default.
• W1921052768 cites W2140983210 @default.
• W1921052768 cites W2146614388 @default.
• W1921052768 cites W2151683280 @default.
• W1921052768 cites W2152141764 @default.
• W1921052768 cites W2157084647 @default.
• W1921052768 cites W2157422578 @default.
• W1921052768 cites W2158787064 @default.
• W1921052768 cites W2160720092 @default.
• W1921052768 cites W2166389083 @default.
• W1921052768 cites W2167078003 @default.
• W1921052768 cites W2168143468 @default.
• W1921052768 cites W308604711 @default.
• W1921052768 doi "https://doi.org/10.1038/emboj.2011.109" @default.
• W1921052768 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3098481" @default.
• W1921052768 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21478823" @default.
• W1921052768 hasPublicationYear "2011" @default.
• W1921052768 type Work @default.
• W1921052768 sameAs 1921052768 @default.
• W1921052768 citedByCount "50" @default.
• W1921052768 countsByYear W19210527682012 @default.
• W1921052768 countsByYear W19210527682013 @default.
• W1921052768 countsByYear W19210527682014 @default.
• W1921052768 countsByYear W19210527682015 @default.
• W1921052768 countsByYear W19210527682016 @default.
• W1921052768 countsByYear W19210527682017 @default.
• W1921052768 countsByYear W19210527682018 @default.
• W1921052768 countsByYear W19210527682019 @default.
• W1921052768 countsByYear W19210527682021 @default.

• next