A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis ´ ´ Macarena Morillo-Huesca, Manuela Vanti and Sebastian Chavez ´ Departamento de Genetica, Universidad de Sevilla, Seville, Spain Keywords gene length; mRNA biogenesis; reporter system; Saccharomyces cerevisiae; transcription elongation Correspondence ´ ´ S Chavez, Departamento de Genetica, Universidad de Sevilla, Facultad de Biologia, Avda ⁄ Reina Mercedes, 6, E41012-Sevilla, Spain Fax: + 34 954557104 Tel: +34 954550920 E-mail: schavez@us.es Enzymes Acid phosphatase (EC 3.1.3.2) (Received 26 September 2005, revised 14 December 2005, accepted 16 December 2005) doi:10.1111/j.1742-4658.2005.05108.x We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene lengthdependent manner We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3¢-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3¢-untranslated regions The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation The use of this assay allows the identification and ⁄ or characterization of new mutants and drugs affecting transcription elongation and other related processes Gene expression is a multistep process involving transcriptional and post-transcriptional events RNA polymerase II-dependent transcription starts by the assembly of the pre-initiation complex (PIC), followed by the initiation step After initiation, transcription elongation is coupled with a set of RNA modifications (capping, splicing and polyadenylation) occurring along the transcription unit Transcription termination is connected to the RNA cleavage required for transcript polyadenylation Formation of mRNP, the mRNA–protein complex transportable to the cytoplasm, is also linked to transcription elongation [1] The traditional view of transcription and RNA processing as separate events has been replaced by mRNA biogenesis as a new concept involving a complex net of functional interactions between RNA processing and the different steps of transcription [2,3] Some elements of the gene expression machinery play a role at the initial steps of mRNA biogenesis whereas some others act all along the transcription unit Among the former, we find the general transcription factors involved in PIC assembly, initiation and early elongation [4,5] The mechanisms of transcriptional regulation of gene expression that take place during these early events have been extensively studied and are fairly well understood [6,7] The latter set of elements is formed by those factors interacting with RNA polymerase II all along transcription elongation Abbreviations GLAM, gene length-dependent accumulation of mRNA; MPA, mycophenolic acid; ORF, open reading frame; PIC, pre-initiation complex; 3¢-UTR, 3¢-untranslated regions; SAGA, Spt-Ada-Gcn5-Acetyltransferase 756 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al [8,9] The relative contribution of this second kind of element to gene expression is expected to depend on the length of the transcription unit, it being greater in longer genes than in shorter ones The distinction between these two types of elements is not always easy when analyzing a mutant or a physiological agent affecting gene expression Nuclear run-on allows measurement of transcription elongation efficiency along the transcription unit [10], but it is timeconsuming and not easy to carry out in a high number of samples In addition, the information obtained by nuclear run-on shows the location of active polymerases, but it gives no information about the quality of the mRNA that is being synthesized This also happens when RNA polymerase II location within the transcription unit is analyzed by chromatin immunoprecipitation; although, in this case, it is possible to distinguish between those elements influencing transcription elongation rate and those involved in processivity [11] Other in vivo assays, such as sensitivity to 6-azauracil or to mycophenolic acid, have been used in yeast to detect transcription elongation defects, and they are easy to perform [12]; however, those assays are too indirect to obtain solid conclusions [13] In vitro assays are useful for defining the role of a given element in a specific step of gene expression (e.g transcription initiation, transcription elongation or splicing), but they are also time-consuming and not recommended as the first assay with which to classify a mutant The best assay for a rapid evaluation of gene expression is the use of a reporter system Available reporter systems allow detection of defects in gene expression, but cannot distinguish between an impairment of the initial steps of transcription, including promoter regulation, and an effect on the subsequent events of mRNA biogenesis We have shown elsewhere that expression of long transcription units in Saccharomyces cerevisiae is more sensitive to mutations affecting the Tho2-Hpr1-Mft1Thp1 (THO) complex [14], connected to transcription elongation and mRNP formation [15] Taking the dependence on the length of the transcription unit as a criterion, we have developed a reporter assay useful for detecting mutations and agents influencing mRNA biogenesis all along the transcription unit In this article, we show that two transcription units sharing the same promoter, terminator and reporter open reading frame (ORF), but differing in the length of their 3¢-untranslated regions (3¢-UTR), are differently influenced by mutations affecting factors that play a role in mRNA biogenesis all along transcription elongation In contrast, those mutations exclusively impairing the initial steps of transcription influence equally the Assay for gene length-dependent mRNA biogenesis expression of the reporters, independently of the length of their 3¢-UTR Results An in vivo assay to measure gene lengthdependent efficiency of mRNA accumulation We constructed several plasmids containing the PHO5 coding region transcribed under the control of the GAL1 promoter, but differing in the length of their 3¢-UTR To increase the length of the 3¢-UTR we inserted Escherichia coli lacZ (either a short fragment or the entire gene in both orientations) or its eukaryotic homolog Kluyveromyces lactis LAC4 These two sequences are equally large but differ in G + C and chromatin organization (see Discussion) Figure 1(A) shows the relative length of the 3¢-UTR of every transcription unit used in this work The ability of the different transcription units to produce full-length mRNA was tested by performing northern analysis on cultures of the respective transformants grown in a galactose-containing medium In all cases, a unique transcript of the expected length was detected (Fig 1B) The intensity of the mRNA signals corresponding to the wild-type cells inversely correlated to the length of the transcription units (see lanes 1–5 in Fig 1B) This result does not necessarily mean that the shortest transcripts (PHO5 alone or PHO5::lacZD) accumulated at a higher level than the longest ones; length also might have influenced the yield of mRNA extraction from the cells and the efficiency of transference during blotting This is likely to be the reason for the variability observed when the results of independent northern experiments are compared (see below) In any case, the quantification of the northern blots indicated that the three longest transcription units showed very similar levels of mRNA accumulation (Fig 1B), despite the sequence of its 3¢-UTR being different (E coli lacZ in both orientations or K lactis LAC4) We hypothesized that those mutations affecting transcription elongation all along the transcription unit should produce a negative length-effect on mRNA accumulation, which was stronger than the one that the wild type might exhibit In order to test this hypothesis, we chose the spt6–140 allele, a thermosensitive mutation affecting a bona fide transcription-elongation factor that colocalizes with RNA polymerase II along the transcription unit [16–18] As shown in Fig 1(B), even at permissive temperature, the signals of the three long transcripts were severely reduced in the mutant strain compared with the wild type, whereas the signals FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 757 Assay for gene length-dependent mRNA biogenesis M Morillo-Huesca et al A B C D Fig Influence of gene-length on mRNA accumulation (A) Transcription units used in this work, corresponding to plasmids pSCh202, pSCh212–18, pSCh212, pSCh211 and pSCh209-LAC4 (B) Northern blot showing the mRNA levels of the five transcription units described in (A) in a wild-type (MMY5.1) and an isogenic spt6–140 strain (MMY5.2) Values were normalized with respect to 25S rRNA Three independent experiments were averaged (C) Relative values of the mRNA levels shown in (B) with respect to the shortest transcript (PHO5 mRNA) (D) GLAM ratios of congenic wild-type and spt6–140 strains: relative levels of acid phosphatase activity expressed by the indicated transcription unit, with respect to the acid phosphatase activity from the shortest transcription unit (PHO5) Averages of four wild-type strains (MMY5.1 ⁄ ⁄ ⁄ 7) and four spt6–140 strains (MMY5.2 ⁄ ⁄ ⁄ 8) are shown For each strain, the average of at least three experiments was considered Error bars indicate standard errors of the short transcripts suffered milder effects To better estimate the effect of length, we calculated the ratios between the mRNA signals of a strain expressing the long transcription units vs the mRNA signal of the same strain expressing just PHO5 (Fig 1C) This long- ⁄ short-transcript ratio was clearly lower in spt6–140 than in the wild type, even when the mRNA containing the 0.3 kb fragment of lacZ was considered the ‘long’ one However, this ratio was dramatically reduced in the mutant when applied to the longest transcription units, being around times lower than in the wild type (Fig 1C) 758 PHO5 encodes a periplasmic acid phosphatase, which is very easy to assay [19] The levels of acid phosphatase activity of an untransformed S cerevisiae strain in SC-galactose medium are almost undetectable, as the endogenous PHO5 gene is repressed in media containing high levels of phosphate [20] As expected, the levels of phosphatase activity in strains lacking our reporter systems were extremely low (not shown) Therefore, the levels of acid phosphatase expressed by our transformants should reflect the amounts of their respective plasmid-encoded PHO5 mRNAs accumulated in the cell Nevertheless, in order FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al to reduce the error caused by the residual expression of the endogenous PHO5, we subtracted the residual acid phosphatase activities of the untransformed strains to all the following results (see Experimental procedures) We assayed acid phosphatase activity in eight congenic strains transformed with our five plasmids: four of the strains being wild-type for SPT6 and four of them having a spt6–140 allele The average of the acid phosphatase activities was used to calculate the ratios between those cells expressing the long transcripts and those expressing the minimal PHO5 mRNA Figure 1(D) shows the calculated ratios When we focused on the wild type, the comparison of the acid phosphatase ratio calculated for PHO5::lacZD to the ratios calculated for the three longest transcription units did not indicate a significant influence of length on Pho5 accumulation As previously suspected, this result suggests that the apparent effect of length on mRNA accumulation in the wild type may be the consequence of a technical bias against long mRNAs during northern experiments Nevertheless, and in agreement with the mRNA ratios shown in Fig 1(C), the acid phosphatase ratios of the spt6 mutant were again clearly lower than those corresponding to the wild type, with the difference being stronger when the long transcript corresponded to any of the three longest 3¢-UTRs (Fig 1D) Moreover, the absolute values of the acid phosphatase ratios are highly reproducible, whereas the mRNA ratios are consistent within a single northern experiment, but show a high variability when comparing different experiments, probably due to differences in mRNA extraction and ⁄ or mRNA transfer during blotting We concluded that measurement of acid phosphatase activity was the best estimation of the mRNA abundance in our systems, and from here on we use the ratio of acid phosphatase activities as an indicator of gene length-dependent accumulation of mRNA (GLAM) Reduced GLAM ratios in transcription-elongation mutants To further confirm that the GLAM ratio is a valid parameter with which to detect defects in mRNA biogenesis, we extended this assay to a set of well-known mutants affected in transcription elongation Three spt16–197 strains, affected in one of the subunits of the yFACT complex [21], clearly showed lower GLAM ratios than three congenic wild types (Fig 2A) The mRNA ratios of a spt16–197 strain and a congenic wild type confirmed the reliability of the phosphatase results (Fig 2B) A similar pattern of GLAM ratios was obtained when comparing an spt4D Assay for gene length-dependent mRNA biogenesis mutant [17,22], lacking one of the subunits of the yDSIF complex, with an isogenic wild type (Fig 2C) Since the length effect was especially clear when using the transcription units containing the longest 3¢-UTR, we chose GAL1pr::PHO5-lacZ and GAL1pr::PHO5LAC4 for the following assays The mRNA ratios calculated for these two transcription units in the spt4D strain and in the isogenic wild type confirmed again the validity of the GLAM ratio to predict elongation defects (Fig 2D) Four additional mutants affected in transcription elongation showed decreased GLAM ratios compared with an isogenic wild type: rpb9D, a 6-azauracil-sensitive mutant lacking a subunit of RNA polymerase II [23]; leo1D and rtf1D, two mutants lacking subunits of the PAF-complex [24,25]; and elp3D, a mutant lacking the histone acetyltransferase subunit of the elongator [26] (Fig 2C) In all these cases, the GLAM ratios of the mutants were, at most, 50% of the wild-type ratios and, therefore, we consider this percentage as the threshold value to indicate a deficiency in gene lengthdependent mRNA biogenesis TFIIS, encoded by the DST1 ⁄ PPR2 gene in S cerevisiae, is a very well known transcription elongation factor involved in releasing RNA polymerase II from arrest sites by stimulating RNA cleavage [27] Although the role of TFIIS in elongation has always been connected to specific pause-sites, and it has never been shown to play a general role in RNA polymerase II-dependent transcription, we performed our assay in a dst1D mutant The GLAM ratios in a dst1D mutant did not show a significant difference with respect to an isogenic wild-type strain when GAL1pr::PHO5-LAC4 was considered the long transcription unit, and only showed a weak decrease when GAL1pr::PHO5-lacZ was chosen (Fig 3A) We repeated the assays in the presence of sublethal doses of the NTP-depleting drug mycophenolic acid (MPA), a drug that has been reported to enhance the transcriptional defects of dst1D mutants by reducing RNA polymerase II processivity [11] As is expected for an inhibitor of transcription elongation, the presence of lgỈmL)1 MPA reduced the GLAM ratio of the wild-type strain (Fig 3A) Similar results were obtained with 6-azauracil (not shown), a drug that also causes depletion of the NTP pools, confirming the suitability of this assay for analyzing elongation-inhibiting drugs However, MPA, even at 10 lgỈmL)1, did not decrease further the GLAM ratio of the dst1D mutant (Fig 3A) As these results separate TFIIS from the other elongation factors studied in this work, we performed northern blot experiments to confirm this negative result based on acid phosphatase data Figure 3(B) shows a FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 759 Assay for gene length-dependent mRNA biogenesis M Morillo-Huesca et al A B C D E Fig Mutants affected in transcription elongation factors show reduced GLAM ratios (A) Averaged GLAM ratios (see legend of Fig 1) of three wild types (MMY11.3 ⁄ ⁄ 12) and three congenic spt16–197 strains (MMY11.4 ⁄ ⁄ 10) (B) Northern blot and mRNA-ratios (see legend of Fig 1) of a wild-type (MMY11.3) and a congenic spt16–197 strain (MMY11.4) Notation of transcription units as in Fig 1(A) (C) GLAM ratios of the spt4D strain Y06986 and the isogenic wild-type BY4741 (D) Northern blot and mRNA ratios of Y06986 and BY4741 E GLAM ratios of rpb9D, leo1D, rtf1D, elp3D and an isogenic wild type (strains Y04437, Y02379, Y04611, Y02742 and BY4741) representative northern experiment which illustrates the absence of a dst1D effect on GLAM; neither the PHO5-lacZ ⁄ PHO5 mRNA nor the PHO5-LAC4 ⁄ PHO5 ratio was affected by dst1D Moreover, the presence of MPA significantly reduced the mRNA levels present in dst1D, but the effect on the long transcription units was proportionally similar to the effect caused on the minimal PHO5 mRNA, therefore producing similar ratios in the wild type and in the mutant strain (Fig 3C) Our results confirm that TFIIS is not playing a general role in mRNA biogenesis all along the transcription unit 760 GLAM ratios are not affected by the impairment of PIC assembly or transcription initiation With the exception of dst1D, all transcription elongation mutants assayed so far showed reduced GLAM ratios One possible explanation for these results is an indirect effect on the GLAM ratios of any major impairment of transcription, regardless of the step of mRNA biogenesis where it occurs To rule out this possibility, we assayed a wide variety of mutants affected in PIC assembly and transcription initiation We analyzed a TBP mutant [28]; a mot1 mutant, affected FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al Assay for gene length-dependent mRNA biogenesis Fig dst1D, even in the presence of mycophenolic acid, does not show reduced GLAM ratios (A) GLAM ratios (see legend of Fig 1) of dst1D and an isogenic DST1 (strains MMY9.2 and BY4741) cultured in the absence or in the presence of sublethal amounts of MPA (B) mRNA levels of the transcription units and strains analyzed in (A) cultured in the absence of MPA The relative values of the mRNA levels with respect to the shortest transcript (PHO5 mRNA) are also shown Three independent experiments were averaged Notation of transcription units as in Fig 1(A) (C) mRNA levels of the transcription units and strains analyzed in (A) cultured in the presence of MPA (10 lgỈmL)1) A representative experiment is shown A B in one of the main TBP regulators [29]; a toa1 mutant, affected in the large subunit of yTFIIA [29]; two sua7 mutants, affected in yTFIIB [30]; two tfa1 mutants, affected in the large subunit of yTFIIE [31]; and two mutants, srb10D and srb11D, lacking subunits of the cyclin–kinase complex that negatively regulates transcription initiation [32,33] None of them showed a significant effect on the GLAM ratios, when compared with the isogenic wild-type strains (Fig 4A–E) Only toa1–18 showed a slightly reduced GLAM ratio when PHO5-lacZ was used as the long transcript, but not when PHO5-LAC4 was considered, excluding a general effect of toa1–18 on GLAM (Fig 4B) Taking together all the results shown in Fig 4, we concluded that the GLAM ratios are not affected by alterations of PIC assembly or transcription initiation Modification of the chromatin structure is an important requirement for transcription regulation and PIC assembly at many promoters One of the main factors involved in this process is the SAGA complex [34], up to now mainly connected to the initial steps of transcription We assayed our reporters in two mutants affecting subunits of SAGA Both gcn5D, a mutant lacking the histone acetyltransferase present in SAGA, and spt3D showed reduced GLAM ratios (Fig 5A) As this result suggests a role of SAGA in mRNA biogenesis all along the transcription unit, we performed northern blot experiments to confirm the acid phosphatase data As shown in Fig 5(B), the accumulation of long mRNAs was more sensitive to the gcn5D mutation than the accumulation of the minimal PHO5 transcript rendering, thus significantly lower ratios One possible explanation for these results is an indirect effect on the GLAM ratios by any mutation generating abnormal chromatin structures In order to test this hypothesis, we analyzed a wide set of mutants including deletions of histone genes like hta1htb1D [35] or htz1D [36]; mutants affected in nucleosome remodeling like isw1D [37], chd1D [38] or swr1D [39]; and an C rpd3D mutant, lacking the main histone deacetylase involved in transcription [40] No significant decrease of the GLAM ratios were observed in any mutant, FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 761 Assay for gene length-dependent mRNA biogenesis M Morillo-Huesca et al A Fig Mutants affected in PIC assembly and transcription initiation not show reduced GLAM ratios GLAM ratios (see legend of Fig 1) of the following mutants and their corresponding wild types: (A) tbp1-P65S and an isogenic TBP1 (strains YAK293 and YAK289) (B) mot1–1, toa1–18 and a wild type with the same genetic background (strains FY1214, JMY498 and FY98) (C) sua7-L50D, sua7K205E and an isogenic SUA7 (strains FP177, FP207 and FP142) (D) tfa1-T218D, tfa1-C127F and an isogenic TFA1 (strains YSB326, YSB331 and YSB324) (E) srb10D, srb11D and an isogenic wild type (strains SLY7, SLY107 and SLY3) B suggesting that the detected effect of the SAGA mutants on the GLAM ratio would not be due to an indirect influence of altered chromatin structure but to a sustained role of SAGA after the initial steps of transcription (Fig 5C–D) Influence of mRNA processing on the GLAM ratios C D E Transcription elongation is coupled with mRNA processing, since all RNA modifications leading to produce a mature exportable mRNA take place or start during elongation We decided to analyze a set of mutations affecting proteins that play a post-transcriptional role in gene expression We included mft1D and thp2D, two mutants lacking subunits of the THO complex [41]; ref2D and syc1D, involved in the 3¢ cleavage previous to polyadenylation and termination [42,43]; and three mutants, cdc40D, cus2D and cwc15D, lacking proteins connected to RNA splicing [44–46] As expected from the reported requirement of the THO complex for the expression of long genes [14], the GLAM ratios of the two THO mutants were dramatically reduced when compared with the wild type (Fig 6) The absence of effect of cdc40D and cus2D on the GLAM ratios was also expected, as no intron is located in the transcription units used in this study However, cwc15D, another mutant connected to splicing, did show a significant reduction (Fig 6) ref2D also showed low GLAM ratios, whereas syc1D, another mutant connected to 3¢ cleavage did not (Fig 6) We conclude that only a subset of mRNAprocessing functions influences mRNA biogenesis in a gene length-dependent manner Discussion Some factors required for mRNA biogenesis play their role during PIC assembly, transcription initiation and early elongation, whereas some others functionally interact with Pol II all along transcription elongation 762 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al A B C D Assay for gene length-dependent mRNA biogenesis Fig Mutants affected in SAGA, but not other chromatin-related mutants, show reduced GLAM ratios (A) GLAM ratios (see legend of Fig 1) of gcn5D, spt3D and an isogenic wild type (strains Y07285, Y04228 and BY4741) (B) Northern blot showing the mRNA levels of the transcription units and strains (gcn5D and wild type) analyzed in (A) The relative values of the mRNA levels with respect to the shortest transcript (PHO5 mRNA) are also shown Notation of transcription units as in Fig 1(A) (C) GLAM ratios of hta1htb1D and an isogenic wild type (strains FY710 and FY120) (D) GLAM ratios of htz1D, swr1D, isw1D, chd1D, rpd3D and an isogenic wild type (strains Y01703, Y03693, Y03385, Y06160, Y01114 and BY4741) Fig Influence of mutations affecting mRNA processing on the GLAM ratios GLAM ratios (see legend of Fig 1) of mft1D, thp2D, cdc40D, cus2D, cwc15D, ref2D, syc1D and an isogenic wild type (strains Y00508, Y02861, Y04201, Y01158, Y03521, Y03554, Y02435 and BY4741) In order to develop an easy test for detecting mutations or drugs that influence mRNA biogenesis all along the transcription unit, we designed a novel two-reporter assay based on the PHO5 gene We hypothesized that gene length is the key element that distinguishes between factors involved in the initial steps of transcription and factors influencing mRNA biogenesis all along the transcription unit, as it is well established for the well-known elongation factor ELL in Drosophila cells [47] We supposed that long transcription units would be more strongly impaired by mutations affecting this second kind of factors than shorter ones, whereas those mutations causing dysfunction of a general factor only involved in the early steps of transcription would affect equally all transcription units, regardless of their length In order to quantify the results of the assay, we have defined gene lengthdependent efficiency of mRNA accumulation as the levels of a long mRNA encoding PHO5, divided by the levels of the minimal PHO5 mRNA, when both FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 763 Assay for gene length-dependent mRNA biogenesis M Morillo-Huesca et al are transcribed from the same promoter We have shown that the so-defined ratio can be estimated by assaying the acid phosphatase activity encoded by PHO5 (GLAM ratio), and that the ratios obtained from acid phosphatase assays are in fact more reproducible than those directly calculated from northern experiments As all transcription units used in this assay express an identical Pho5 protein, it is unlikely that mutations or drugs affecting translation, protein stability or other post-translational processes, may influence the GLAM ratios We tested the consistency of the GLAM ratios by performing the assay in a wide set of previously characterized mutants involved in mRNA biogenesis The GLAM ratios were always calculated for two different long transcription units: the one containing E coli lacZ (GAL1pr::PHO5-lacZ) and the other containing K lactis LAC4 (GAL1pr::PHO5-LAC4) These two genes share the same length, but display a very different G + C content Their chromatin structure in S cerevisiae is also completely different: random nucleosome positioning in lacZ [14] but translationally posi´ tioned nucleosomes in LAC4 (S Jimeno-Gonzalez, ´ P M Alepuz and S Chavez, unpublished) We considered that a mutant showing similar GLAM ratios with both long transcription units would indicate a gene length-dependent effect, whereas a mutant exhibiting differences between the GLAM ratios calculated with each long transcript might involve sequence-dependent or chromatin-dependent phenomena Among all the mutants analyzed in this study, only toa1–18 affecting TFIIA showed a significant difference between the GLAM ratio calculated with GAL1pr::PHO5-LAC4 and the one calculated with GAL1pr::PHO5-lacZ We did not find an explanation for this result, unless a connection exists between TFIIA and the chromatin organization of the transcribed region In all other strains tested, the two values were not significantly different, although in most cases the GLAM ratios calculated with GAL1pr::PHO5-lacZ were slightly higher than those calculated with GAL1pr::PHO5-LAC4 Mutations affecting transcription elongation factors, such as SPt6, yFACT, yDSIF, and the PAF1 complex, as well as the elongator, clearly showed lower GLAM ratios than their isogenic wild types In contrast, those mutations described to affect factors involved in PIC assembly or transcription initiation, like TBP, Mot1, TFIIB, TFIIE or Srb10-Srb11, show very similar GLAM ratios compared with their corresponding wild-types, with SAGA mutations being the only exception (discussed below) In all these mutants, the resulting GLAM ratios for two long transcription units (GAL1pr::PHO5-lacZ and GAL1pr::PHO5-LAC4) 764 were very similar Moreover, the presence of sublethal doses of nucleotide-depleting drugs affecting transcription elongation also reduced the GLAM ratios We conclude that these general results are solid enough to validate the novel two-reporter assay as a useful tool with which to detect transcription elongation defects Mutations or drugs can affect two different aspects of transcription elongation: elongation rate and processivity A recent study by Mason and Struhl [11] has shown that none of the many putative elongation factors that they tested affect the elongation rate, although mutations in the THO complex and Spt4 significantly reduce processivity Our assay is based on the comparison of transcription units of different length, which makes it an optimal method with which to detect processivity defects; consequently thp2, mft1D and spt4D show the lowest GLAM ratios Mason and Struhl also showed that those elements affecting elongation rate, such as 6-azauracil and MPA, simultaneously reduce processivity [11]; the decrease of the GLAM ratios in response to these two NTP-depleting drugs indicates that our in vivo assay can detect all the elongation defects detected by RNA polymerase IIdependent chromatin immunoprecipitation Moreover, some putative elongation mutants that did not show reduced processivity in the study by Mason and Struhl, such as elp3D, rtf1D and leo1D [11], did show significantly low GLAM ratios, suggesting that our in vivo assay displays a high sensitivity to detect elongation defects Finally, mutations in SPt6 and SPt16 that cannot be analyzed by RNA polymerase II-dependent chromatin immunoprecipitation due to technical limitations of that assay [11], show reduced GLAM ratios, supporting a contribution of FACT and SPt6 to processivity We conclude that the new in vivo assay described in this study is a convenient complementary tool with which to analyze transcription elongation The only transcription elongation factor tested whose mutation did not produce lower GLAM ratios than its isogenic wild type was TFIIS When either calculated with the assayed acid phosphatase activities or inferred from northern experiments, the ratios of a dst1D mutant were not significantly low Moreover, the presence of sublethal doses of nucleotide-depleting drugs, like 6-azauracil or MPA, reduced the GLAM ratios of a dst1D mutant; however, it also reduced the GLAM ratios of an isogenic wild type accordingly This differentiated behavior of dst1D separates TFIIS from the other transcription elongation factors tested in this work The most logical explanation for this phenomenon is that, as has been recently suggested [11], TFIIS does not play a relevant role in elongation all along the transcription unit, or at least along the FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al transcription units used in this assay Alternately, TFIIS may play a general function, but only during early elongation If this were the case, both long and short transcription units would be equally influenced by the absence of TFIIS, and as a result the GLAM ratio would be unaffected A role of TFIIS centered in early elongation has been suggested by the genetic and physical interactions of TFIIS [48,49] A relevant role of TFIIS in early elongation has been also found in Drosophila heat-shock genes [50] Moreover, a role of TFIIS in activating the GAL1 promoter has been recently demonstrated [51] An elongation role of TFIIS in vivo, in positions far from the promoter, has only been shown when an artificial arrest site was introduced, and even in this case the influence of TFIIS depended on the degree of transcriptional activation [52] The present results confirm the difficulties involved in studying the dst1D mutant in vivo, an observation already reported elsewhere [11] The SAGA complex is one of the main elements that mediate in activation of gene expression; it acts through its ability to interact with gene-specific factors and to stimulate PIC assembly Surprisingly, gcn5D and spt3D mutants, both lacking subunits of SAGA, show reduced GLAM ratios We consider it unlikely that these gene length-dependent effects of the SAGA mutants take place at the level of PIC assembly, since all transcription units used to calculate the GLAM ratios share the same promoter We have also ruled out an indirect effect of SAGA mutations on the GLAM ratios provoked by overall effects on chromatin structure, just as other mutations affecting structural elements of chromatin (hta1htb1D, htz1D) or chromatin remodeling (swr1D, isw1D, chd1D, rpd3D) show similar GLAM ratios to their isogenic wild types We conclude that SAGA might play an additional role after transcription initiation all along the transcription unit The genetic interactions of GCN5 with the elongator [53], the sensitivity of several SAGA mutants to mycophenolic acid [54] and the genetic interactions between genes encoding SAGA subunits and elongation factors [55,56] also suggest a role for SAGA during transcription elongation Alternatively, the absence of SAGA might affect the recruitment of other factors required for postinitiation events in mRNA biogenesis As discussed above, mutants affected in factors required for transcription elongation all along the transcription unit show reduced GLAM ratios However, transcription elongation is not the only gene-length dependent process in mRNA biogenesis Formation of the mRNP complex, mRNA export or splicing are other events that may be gene lengthdependent Mutants lacking subunits of the THO Assay for gene length-dependent mRNA biogenesis complex, involved in mRNP formation, show the lowest GLAM ratios measured in this work This is not the case in other RNA processing mutants that have been analyzed in this work In agreement with our results, an important part of the RNA-processing machinery does not significantly influence mRNA accumulation [15] However, a part of the RNA processing machinery physically interacts with elongating Pol II; as a consequence, the absence of elements involved in RNA processing may indirectly affect transcription elongation [57] This might be the explanation of the low GLAM ratios shown by cwc15D and ref2D, lacking proteins connected to splicing and 3¢ cleavage, respectively In contrast to the consistency of the results obtained using our reporter assay with well-known mutants affected in transcription initiation or elongation, the behavior of mutations affecting mRNA processing is heterogeneous Additional analyses are required to understand more fully the effect of these mutations on gene length-dependent accumulation of mRNA Experimental procedures Materials Suppliers are indicated below at first mention, except for chemical reagents, which were purchased from Sigma (St Louis, MO, USA) Yeast strains, plasmids and media Yeast strains used are described in Table All MMY strains were constructed by standard genetic methods of tetrad analysis or transformation [58] MMY5 strains are congenic and were generated by crossing FY120 and FY137 AGY1–10 A strain was obtained by crossing FY348 and W303-ZT; further crossing of FY120 and AGY-10A rendered all MMY11 strains MMY9.2 was obtained by sporulating Y24411 All plasmids used are mono-copy CEN-based and are listed in Table Cells were grown in yeast extract–peptone medium or in synthetic complete medium (DIFCO, Detroit, MI, USA), with 2% glucose or 2% galactose, at 30 °C [58] Acid phosphatase assays Yeast cells with the appropriated plasmids were grown on selective synthetic medium lacking uracil with 2% galactose and collected when cultures reached an optical density at 600 nm (OD 600) of 0.8–1 Acid phosphatase activity of intact cells was assayed as described [19] The acid phosphatase activities of the transformants were corrected by FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 765 Assay for gene length-dependent mRNA biogenesis M Morillo-Huesca et al Table Strains Strain Genotype Source FP142 FP177 FP207 FY120 FY1214 FY137 FY348 FY710 FY98 JMY498 SLY107 SLY3 SLY7 W303-ZT YAK289 YAK293 YSB324 YSB326 YSB331 BY4741 Y00508 Y01114 Y01158 Y01586 Y01703 Y02379 Y02435 Y02742 Y02861 Y03385 Y03521 Y03554 Y03693 Y04201 Y04228 Y04437 Y04611 Y06160 Y06986 Y07285 Y24411 AGY1–10A MMY5.1 MMY5.2 MMY5.3 MMY5.4 MMY5.5 MMY5.6 MMY5.7 MMY5.8 MMY9.2 MMY11.3 MMY11.4 MMY11.6 MMY11.8 MMY11.10 MMY11.12 MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ SUA7] MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ sua7-L50D] MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ sua7-K205E] MATa leu2D1 ura3 his4–912d lys2–128d MATa leu2D1 ura3–52 mot1–1 MATa ura3 his4–912d lys2–128d spt6–139 MATa leu2D1 ura3 his4–912d lys2–128d spt16–196 MATa leu2D1 ura3 his4–912d lys2–128d hta1-htb1::LEU1 MATa leu2D1 ura3–52 MATa ura3–52 his4–912d lys2–128d toa1–18GSG MATa his3D200 leu2–3,112,ura3–52 srb11D1::hisG MATa his3D200 leu2–3,112,ura3–52 MATa his3D200 leu2–3,112,ura3–52 srb10D1::hisG MATa leu2 his3 ade2 trp1 ura3::GAL-lacZ::URA2 MATa ura3–52 trp1–63 leu2,3–112 his3–609Dspt15[pTM8 ⁄ TBP] MATa ura3–52 trp1–63 leu2,3–112 his3–609Dspt15[pTM1228 ⁄ TBP-P65S] MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK6 ⁄ TFA1] MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK1DSpe] MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK6 ⁄ tfa1-C127F] MATa his3D1 leu2D0 met15D0 ura3D0 MATa his3D1 leu2D0 met15D0 ura3D0 mtf1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 rpd3::KAN MATa his3D1 leu2D0 met15D0 ura3D0 cls2::KAN MATa his3D1 leu2D0 met15D0 ura3D0 snf2::KAN MATa his3D1 leu2D0 met15D0 ura3D0 htz1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 leo1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 syc1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 elp3::KAN MATa his3D1 leu2D0 met15D0 ura3D0 thp2::KAN MATa his3D1 leu2D0 met15D0 ura3D0 isw1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 cwc15::KAN MATa his3D1 leu2D0 met15D0 ura3D0 ref2::KAN MATa his3D1 leu2D0 met15D0 ura3D0 swr1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 cdc40::KAN MATa his3D1 leu2D0 met15D0 ura3D0 spt3::KAN MATa his3D1 leu2D0 met15D0 ura3D0 rpb9::KAN MATa his3D1 leu2D0 met15D0 ura3D0 rtf1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 chd1::KAN MATa his3D1 leu2D0 met15D0 ura3D0 spt4::KAN MATa his3D1 leu2D0 met15D0 ura3D0 gcn5::KAN MATa ⁄ a his3D1 ⁄ his3D1 leu2D0 leu)2D0 lys2D0 ⁄ LYS2 MET15 ⁄ met15D0 ura3D0 ⁄ ura3D0 dst1::KAN ⁄ DST1 MATa ade2 trp1leu2 his4–912d lys2–128d ura3::GAL1-lacZ::URA3 spt16–197 MATa ura3 his4–912d lys2–128d MATa ura3 his4–912d lys2–128d spt6–140 MATa ura3 his4–912d lys2–128d leu2 spt6–140 MATa ura3 his4–912d lys2–128d leu2 MATa ura3 his4–912d lys2–128d spt6–140 MATa ura3 his4–912d lys2–128d MATa ura3 his4–912d lys2–128d leu2 MATa ura3 his4–912d lys2–128d leu2 spt6–140 MATa his3D1 leu2D0 met15D0 ura3D0 dst1::KAN MATa trp1leu2 his4–912d lys2–128d ura3 MATa ade2 trp1 his4–912d lys2–128d ura3 spt16–197 MATa ade2 leu2 his4–912d lys2–128d ura3 spt16–197 MATa ade2 leu2 his4–912d lys2–128d ura3 MATa trp1leu2 his4–912d lys2–128d ura3 spt16–197 MATa leu2 his4–912d lys2–128d ura3 [30] [30] [30] [17] [29] [17] [59] [35] [29] [29] [32] [32] [32] J Svejstrup laboratory [28] [28] [31] [31] [31] EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 766 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS M Morillo-Huesca et al Assay for gene length-dependent mRNA biogenesis Table Plasmids Plasmid Description Source or reference YCplac33 pSCh202 pSCh212 pSCh211 pSCh212–18 pSCh209-LAC4 YCp vector based on the URA3 gene pRS416 containing the PHO5 region fused to the GAL1 promoter pSCh202 with lacZ transcriptionally fused to the 3¢-end UTR of PHO5 pSCh212 with lacZ in the opposite orientation pSCh202 with a 0.3-kb fragment of the 3¢-end of lacZ fused to the 3¢-end UTR of PHO5 pSCh202 with K lactis LAC4 transcriptionally fused to the 3¢-end UTR of PHO5 [60] [61] [14] [14] [14] This study subtracting the residual acid phosphatase activity of the corresponding untransformed strain Northern analyses Six micrograms of total RNA prepared from yeast cells, collected in the conditions described above, were subjected to electrophoresis on formaldehyde-agarose gels (agarose form Pronadisa, Madrid, Spain), transferred to Hybond-N filters (Amersham Biosciences UK, Buckinghamshire, UK), and UV cross-linked prior to hybridization at 65 °C in 0.5 m sodium phosphate buffer, pH 7, 7% SDS, with a [32P]dCTP-labeled DNA PHO5 probe Quantification of mRNA levels was performed in a Phosphorimager All values were normalized with respect to the present amount of 25S rRNA, detected by hybridization with a [32P]-oligolabeled 589 bp rDNA internal PCR fragment amplified with the 19-mer oligonucleotides 5¢-TTGGAGAGGGCA ACTTTGG-3¢ and 5¢-CAGGATCGGTCGATTGTGC-3¢ (Stab Vida, Oeiras, Portugal) Acknowledgements ´ We thank Francisco Malagon and Francisco Navarro for their critical reading of the draft; we also thank S Buratowski, T Kokubo, A Ponticelli, J Svejstrup, F Winston and R Young for strains and plasmids, and Antonio Garcı´ a-Susperregui for strains construction This work 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the best estimation of the mRNA abundance in our systems, and from here on we use the ratio of acid phosphatase activities as an indicator of gene. .. tested the consistency of the GLAM ratios by performing the assay in a wide set of previously characterized mutants involved in mRNA biogenesis The GLAM ratios were always calculated for two... caused on the minimal PHO5 mRNA, therefore producing similar ratios in the wild type and in the mutant strain (Fig 3C) Our results confirm that TFIIS is not playing a general role in mRNA biogenesis