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• W2044346282 abstract "Article3 February 2005free access Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins Dipayan Rudra Dipayan Rudra Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Yu Zhao Yu Zhao Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Jonathan R Warner Corresponding Author Jonathan R Warner Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Dipayan Rudra Dipayan Rudra Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Yu Zhao Yu Zhao Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Jonathan R Warner Corresponding Author Jonathan R Warner Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Author Information Dipayan Rudra1, Yu Zhao1 and Jonathan R Warner 1 1Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA *Corresponding author. Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Tel.: +1 718 430 3022; Fax: +1 718 430 8574; E-mail: [email protected] The EMBO Journal (2005)24:533-542https://doi.org/10.1038/sj.emboj.7600553 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5–10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus. Introduction With the recent addition of Asc1p (Link et al, 1999), we now know that the Saccharomyces cerevisiae ribosome has 79 proteins, encoded by 138 ribosomal protein (RP) genes that are responsible for nearly 50% of all Pol II transcriptional initiations (Velculescu et al, 1997; Holstege et al, 1998; Warner, 1999). Their transcription is rigorously controlled as a cohort in response to both positive and negative signals (Gasch et al, 2000; Causton et al, 2001). Indeed, they represent the most prominent cluster in most transcriptome studies. The transcriptional activating regions of most RP genes are characterized by a pair of sites that bind Rap1p and are essential for high-level transcription (Rotenberg and Woolford, 1986; Schwindinger and Warner, 1987; Nieuwint et al, 1989). In a few cases, the Rap1p sites are replaced by a site for Abf1p (Hamil et al, 1988; Herruer et al, 1989) or for Reb1p (Lascaris et al, 1999). The coordinate regulation of transcription of the RP genes appears independent of which transcriptional regulator is present. Rap1p is a protein of many functions, as its name, repressor-activator-protein, suggests (reviewed in Morse, 2000; Pina et al, 2003). It is responsible for the transcription not only of the RP genes, but also of many genes encoding translation factors and enzymes of glycolysis. It binds to the TG repeats of telomeric DNA. It serves to nucleate complexes that repress transcription of genes both adjacent to the telomeres and at the silent MAT loci. Yet alone Rap1p has only weak transcriptional activating ability (Tornow et al, 1993). It is reported to act by interfering with nucleosomes, thus facilitating the access of activation factors to their binding sites (Yu and Morse, 1999). Although acetylated histones have been observed throughout the UAS of RP genes (Reid et al, 2000), recent reports suggest that the promoter regions of actively transcribed genes, especially RP genes, are nearly devoid of nucleosomes (Bernstein et al, 2004; Lee et al, 2004). The specificity of Rap1p in its several roles presumably lies in its recruitment of specific coactivators, such as Gcr1p at the glycolytic genes (Tornow et al, 1993), or corepressors, such as Sir3p, at telomeres and silent MAT loci (Moretti et al, 1994). Recently, a genome-wide chromatin immunoprecipitation (ChIP) analysis showed that the promoter of nearly every RP gene is occupied by the hitherto obscure potential transcription factor Fhl1p (forkhead-like) (Lee et al, 2002). Fhl1p was originally identified as a multicopy suppressor of a Pol III mutant, and was then shown to be important for ribosome biosynthesis (Hermann-Le Denmat et al, 1994). Subsequently, the same group identified IFH1 as a multicopy suppressor of the slow growth phenotype of a ΔFHL1 strain. Ifh1p, essential for growth, was also implicated in ribosome biosynthesis. Surprisingly, cells with deletions of both FHL1 and IFH1 survive (Cherel and Thuriaux, 1995). We have now explored in more detail both the roles of Fhl1p and Ifh1p in ribosome biosynthesis and the physiological effects of their absence. We confirm that Fhl1p, as well as Ifh1p, is found at the UAS of RP genes. By co-immunoprecipitation (Co-IP) analysis, we find that Fhl1p and Ifh1p interact with each other through the ‘forkhead (FH)-associated' (FHA) domain of Fhl1p (Durocher and Jackson, 2002). Mutation of the FHA domain, reducing its interaction with Ifh1p, leads to loss of Ifh1p from RP genes and to severe defects in ribosome synthesis and growth. Treatment of cells with rapamycin, which represses strongly the transcription of RP genes (Cardenas et al, 1999; Powers and Walter, 1999), leads to the loss of Co-IP of Fhl1p with Ifh1p and to the disappearance of Ifh1p from the RP genes. Together, these observations suggest that the Fhl1p–Ifh1p interaction is responsible for active transcription of the RP genes. Cells lacking Fhl1p or both Fhl1p and Ifh1p grow exceedingly slowly and have less than one-quarter the normal amount of ribosomes, presumably because of deficient transcription of RP genes. Nevertheless, these ribosome-deprived cells utilize homeostatic mechanisms both to reduce their transcription of rRNA to match the available RPs and to balance their total mRNA population to the available ribosome complement. Results Both Fhl1p and Ifh1p are associated with RP genes To confirm and extend the results reported by Lee et al (2002), we performed ChIP analysis on a strain carrying Fhl1p C-terminally tagged with HA3 and Ifh1p C-terminally tagged with Myc9. As shown in Figure 1A (lanes 4 and 6), ChIP with either anti-HA or anti-Myc enriched for DNA fragments from the promoter regions of RP genes, RPL3, RPL7A, RPL28, RPL30 and RPS6A. No such enrichment was seen for promoters of PGK1, which is also driven by Rap1p (Packham et al, 1996), or for ACT1. Parallel analysis of an untagged strain showed no enrichment (lanes 3 and 5). Figure 1.Fhl1p and Ifh1p are associated with RP gene promoters. (A) ChIP was performed using anti-HA or anti-Myc antibodies on W303a (WT) and DR36 (FHL1-HA3, IFH1-MYC9) double-tagged strains. Following IP, PCR was performed on total chromatin (input) and the immunoprecipitated (IP) DNA with primers specific for the promoters of the indicated RP genes. Primers specific for the promoters of non-RP genes PGK1 and ACT1 were used as controls. (B) A real-time PCR performed on the samples from strain DR36 (FHL1-HA3, IFH1-MYC9) in (A) using primers for the promoters of the indicated genes. Calculation of the ‘fold enrichment’ values is documented in Materials and methods. (C, D) FHL1-HA3, IFH1-MYC9 (strain DR47) double-tagged cells were pretreated for 30 min with rapamycin or the drug vehicle DMSO prior to formaldehyde crosslinking. This was followed by ChIP using anti-HA (C) or anti-Myc (D) antibodies followed by real-time PCR analysis. Download figure Download PowerPoint Quantitative PCR analysis of ChIP products (Figure 1B) showed a 10- to 20-fold enrichment of Fhl1p and a five- to eight-fold enrichment of Ifh1p at the promoters of several RP genes, with a lesser, but reproducible, enrichment at other RP genes. These results show that both Fhl1p and Ifh1p can be found at RP promoters. Their presence at RP gene promoters cannot depend on Rap1p alone as RPL3 has a single Abf1p site rather than two Rap1p sites (Hamil et al, 1988). Loss of Ifh1p from the RP genes during repression Rapamycin leads to a rapid reduction in transcription of rRNA and RP genes (Cardenas et al, 1999; Powers and Walter, 1999). ChIP analysis shows that after treatment of the cells with rapamycin, Fhl1p nevertheless remains at the promoters of the RP genes (Figure 1C). On the other hand, Ifh1p does not (Figure 1D). This result suggests that the presence of Ifh1p is associated with the activation of transcription of the RP genes. Note that Rap1p is constitutively bound to RP promoters, whether transcription is occurring or not (Reid et al, 2000) as we have confirmed (data not shown). Thus, the repression of RP gene transcription due to rapamycin is accompanied by the loss of Ifh1p from the RP genes. Ifh1p acts as a regulator of RP genes The observation that the rapid transcription of RP genes is coincident with their occupancy by Ifh1p, but not Rap1p or Fhl1p, suggests that Ifh1p is an important regulator. To test this notion, we generated a strain with IFH1 under control of the GAL1 promoter (GALUAS-IFH1). Although deletion of IFH1 is lethal (Cherel and Thuriaux, 1995), these cells grow slowly on the limiting amount of Ifh1p synthesized under glucose repression, while in the presence of galactose they grow comparably to wild-type (WT) cells (Figure 2A). Although Ifh1p is initially below detection, it is rapidly synthesized after the culture is shifted from glucose to galactose (Figure 2B). The appearance of Ifh1p is accompanied by a rapid increase in transcription of RP genes, without much change to the levels of non-RP mRNAs derived from ACT1 or TEF1 (Figure 2C). This result suggests that limiting Ifh1p leads to limiting transcription of RP genes. Figure 2.Ifh1p as a regulator of RP genes. (A) Growth of YZ146 (IFH1-HA3 (WT)) and YZ147 (GALUAS-HA3-IFH1) in glucose (YPD) and galactose (YPGal) respectively. Cultures of WT (YZ146) and GALUAS-HA3-IFH1(YZ147) cells were grown in YPD media, and were shifted to YPGal by filtering. Cells were harvested at the indicated time points. (B) A portion was prepared for Western analysis using antibodies directed against the HA epitope or against Rap1p. (C) From the rest, RNA was prepared and Northern analysis was performed to determine the level of the indicated mRNAs by normalizing with the U3 snoRNA. A graphical representation of the ratio (GAL-IFH1/WT) at each time point is shown. Note that the total RNA level of these cells when grown in glucose is only 1/5 that of WT cells. As will be discussed below (Figures 7 and 8), limiting availability of Ifh1p leads to a downregulation of total RNA as well as of all mRNAs to match the availability of the translational apparatus. The levels of U3 snoRNA remain relatively constant. Thus when normalized against U3 snoRNA, at the 0 time points the ratio of both RP and non-RP mRNAs in mutant versus WT cells in glucose medium is approximately 0.2. Download figure Download PowerPoint Neither Fhl1p nor Ifh1p binds to RP promoters in vitro In an attempt to dissect the system, we carried out band-shift experiments using the intergenic region upstream of the RP gene, RPL11A, together with partially purified TAP derivatives (Puig et al, 2001) of Fhl1p and Ifh1p, alone, together and with Rap1p. While Rap1p binds tightly, no evidence of binding by Fhl1p or Ifh1p was observed, alone or in combination with the others, using several concentrations of the proteins (Figure 3). Similar results were obtained using sequences upstream of RPL28 and RPL30 (data not shown). By contrast, other ‘FH’ proteins of yeast, Fkh1p and Fkh2p, bind to multiple targets (Hollenhorst et al, 2001), sometimes with the assistance of other proteins (Kumar et al, 2000). The lack of direct binding by Fhl1p and by Ifh1p suggests that they require other factors or specific chromatin structures to associate with the RP gene promoters. Figure 3.Failure of Fhl1p and Ifh1p to bind an RP promoter. A radiolabeled PCR-amplified fragment encompassing the intergenic region between RPL11A and PRE2 (1 ng) was mixed with partially purified TAP-tagged Rap1p (5–10 ng), Fhl1p and Ifh1p (50–100 ng), or mock-purified product from an untagged strain, either separately or together as indicated in the figure, for 60 min at 0°C in 20 μl of solution containing 20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 0.5 mM DTT, 5% glycerol, 5 μg poly(dI-dC), 20 μg BSA and 2 mM PMSF. Nondenaturing polyacrylamide gel electrophoresis on 8% acrylamide gels run in 25 mM Tris–borate and 0.25 mM EDTA to resolve any DNA–protein complex formed was followed by autoradiography of the dried gel. Download figure Download PowerPoint Fhl1p and Ifh1p interact with each other We carried out Co-IP experiments to ask if the genetic interaction of Fhl1p and Ifh1p (Cherel and Thuriaux, 1995) arises from a physical interaction between the two proteins. As shown in Figure 4A, HA-tagged Fhl1p will co-immunoprecipitate Myc-tagged Ifh1p (lane 3); conversely, Myc-tagged Ifh1p will co-immunoprecipitate HA-tagged Fhl1p (Figure 4B, lane 5). No IP was observed in untagged strains (Figure 4A, lane 4; Figure 4B, lane 5). The Co-IP is not mediated through common interaction with DNA, because it is unaffected by the presence of ethidium bromide, which intercalates into DNA, thereby inhibiting normal protein–DNA interactions (Lai and Herr, 1992) (Figure 4B, lane 7). In cells pretreated with rapamycin, the interaction between Fhl1p and Ifh1p is greatly diminished (Figure 4C, compare lanes 3 and 4). Thus, rapamycin leads to the repression of RP gene transcription, the loss of Ifh1p from the RP genes and sharply reduced interaction between Fhl1p and Ifh1p. It seems likely that the interaction between Fhl1p and Ifh1p is a cause of the high level of transcription of RP genes. Figure 4.Fhl1p and Ifh1p interact with each other. (A) Co-IP was carried out using anti-HA antibody on extracts prepared from DR36 (FHL1-HA3, IFH1-MYC9 double-tagged), with DR37 (IFH1-MYC9) as a negative control. The immunoprecipitated protein complex was resuspended in SDS loading buffer, boiled and analyzed by SDS–PAGE followed by Western blotting using anti-HA or anti-Myc antibodies. A 5 μl portion of the original cell extracts was analyzed in separate lanes as loading controls (input). (B) A converse Co-IP experiment to Figure 2A. In this case, the IP was carried out using anti-Myc antibody on extracts prepared from DR36 (FHL1-HA3, IFH1-MYC9), with DR13 (FHL1-HA3) as a negative control. Samples in the lanes indicated were treated with 200 μg/ml of ethidium bromide for 30 min on ice before the IP (see Materials and methods). (C) Extracts of DR36 (FHL1-HA3, IFH1-MYC9) cells that had been treated with 0.2 μg/ml rapamycin or with drug vehicle (DMSO) for 30 min were subjected to Co-IP using anti-Myc antibody. Download figure Download PowerPoint Basis of the interaction of Fhl1p with Ifh1p Examination of the sequence of Fhl1p reveals two conserved domains (Figure 5A). FH has been identified as a DNA-binding domain, unusual in that it requires Mg ions for binding (Clark et al, 1993). The FHA domain is widespread in nature, originally identified in FH proteins, but now observed in many others. The FHA domain binds to distinct phospho-peptide ligands, usually those containing a phospho-threonine (reviewed in Durocher and Jackson, 2002). By contrast, the 1085-amino-acid sequence of Ifh1p has no easily recognizable domains. Figure 5.The FHA domain of Fhl1p is required for its interaction with Ifh1p. (A) Schematic of the Fhl1p protein with the FHA domain (amino acids 300–374) and the FH domain (amino acids 440–567) indicated. (B) Co-IP using anti-Myc antibody on extracts prepared from equal numbers of cells of strains DR47 (FHL1-HA3), DR48 (ΔFH-HA3) or DR49 (ΔFHA-HA3), carried on a CEN plasmid, covering the deleted FHL1; Ifh1p is tagged C-terminally with Myc9. (C) Cultures were grown in synthetic media at 30°C with gentle shaking and the growth rate determined over several generations by light scattering at 600 nm. The mutations in FHL1 are indicated. The Mg2+ site mutant consisted of the following changes: L514A, S515A, N517A and F520A. Deletion of the FH domain includes amino acids 440–567 and that of the FHA domain includes amino acids 300–374. The total RNA isolated from 1 ml of a culture of W303a at OD600∼1.0 is arbitrarily defined as 1 unit. The relative amount of RNA from the indicated strains at a similar optical density is tabulated. ND: not done. (D) A 7.5 μg portion of RNA isolated from the indicated strains (requiring 5 × as many mutant as WT cells) was mixed with ethidium bromide and analyzed on a denaturing agarose gel and photographed under UV illumination. (E) WT and FHL1 mutant strains viewed at 100 × magnification with Nomarski optics. Download figure Download PowerPoint We generated mutant versions of Fhl1p in order to determine the sites of interaction with Ifh1p. Several mutations within the FH domain or deletion of the entire FH domain has no effect on the interaction (Figure 5B, lane 7). On the other hand, deletion of the FHA domain of Fhl1p, or even mutation of a single amino acid within the core of the FHA domain, S325 to R, leads to loss of interaction with Ifh1p (Figure 5B, lanes 6 and 8). Since the FHA domain is known to interact with a phospho-peptide, this result predicts that transcription of RP genes is related to the phosphorylation of a site on Ifh1p that leads to its interaction with the FHA domain of Fhl1p. Interaction of Fhl1p and Ifh1p is important for cell growth Deletion of FHL1, while not lethal, reduces substantially the growth rate of a cell (Hermann-Le Denmat et al, 1994) (Figure 5C). Furthermore, the RNA content of such cells is markedly decreased (Figure 5C). As is evident from Figure 5D, which employed RNA from five times as many mutant as WT cells, this decrease is largely in rRNA. Quantitation of Figure 5D indicates that deletion of FHL1 reduces the mass ratio of rRNA to tRNA from 4.8 to 0.6. Stated another way, mutant cells have only 10% the number of ribosomes but 70% the number of tRNAs of WT cells, substantiating the conclusion that Fhl1p is important for ribosome synthesis. However, neither mutation of key residues within the FH domain nor deletion of the entire domain had much effect either on cell growth or on RNA content (Figure 5C). By contrast, deletion of the FHA domain caused nearly as slow growth, and nearly as reduced RNA, as did deletion of the entire gene. The mutation S325R of FHL1, shown above to reduce the interaction of Fhl1p with Ifh1p, has an intermediate effect on growth and on ribosome content (Figure 5C). Note that the mutant forms of Fhl1p are present at the same level as the WT protein (Figure 5B, inputs). The morphology of the cells is shown in Figure 5E. Although the appearance of unnatural protrusions suggests problems in cell division, the key observation is that in each strain, the cells are roughly the same size as WT cells. This contrasts with the unusually small size of cells deficient in Sfp1p, another factor implicated in ribosome synthesis (Jorgensen et al, 2004). Occupancy of the RP promoter by Ifh1p requires its interaction with Fhl1p The consistency of the observations in vivo and in vitro leads to the hypothesis that a key step in driving the transcription of RP genes is the binding of Ifh1p to Fhl1p, rather than the binding of Fhl1p to DNA (or to chromatin). We carried out a ChIP experiment to determine the occupancy of Fhl1p and Ifh1p at RP promoters under conditions in which they do not interact with each other. Neither deletion of the FHA domain nor the mutation S325R affects the association of Fhl1p with RP genes (Figure 6A). Strikingly, however, deletion of the FHA domain abolishes the association of Ifh1p with the RP genes, and mutation S325R nearly does so (Figure 6B). Taken together, these results indicate that it is the interaction between Fhl1p and Ifh1p that is responsible for bringing Ifh1p to the RP promoters, and suggest that while the presence of Fhl1p seems to be the unique characteristic of RP genes (Lee et al, 2002), it is the presence of Ifh1p that leads to their active transcription. Figure 6.Interaction of Fhl1p and Ifh1p is necessary to bring Ifh1p to the RP genes. A ChIP experiment followed by real-time PCR was performed using anti-HA (A) or anti-Myc (B) antibodies on strains harboring HA3-tagged full-length (WT), FHA domain deleted (ΔFHA) or the S325R mutant version of Fhl1p (strains DR47, DR49 and DR65, respectively). The endogenous copy of FHL1 in these strains is deleted, and Ifh1p is tagged C-terminally with Myc9. Download figure Download PowerPoint Physiology of ribosome-deprived cells In spite of the critical role that Fhl1p and Ifh1p play in ribosome synthesis, cells with the genotype ΔFHL1 or ΔFHL1 ΔIFH1 are viable, if very slow growing (Cherel and Thuriaux, 1995). While the slow growth is presumably due to the reduced content of ribosomes, how do the cells manage their transcription and translation under such conditions? We determined the level of rRNA transcription and the efficiency of rRNA processing by pulse–chase labeling with [C3H3]-methionine, with which it is possible to look specifically at the rRNA species (Figure 7). Two results are apparent. First, the level of incorporation of CH3 during the 2.5 min pulse is greatly reduced in each mutant strain. Second, the processing of the pre-rRNA appears to proceed slowly but relatively efficiently in each mutant strain. This result is more apparent in the right-hand panels, which show a longer pulse and a longer chase for the mutant strains. By far, the larger part of the precursor RNAs appears to be processed normally, although there is a suggestion of some degradation. Since the mutant cells have a doubling time 3–4 × greater than the WT cells, and only 1/5 the content of RNA (Figure 5C), we calculate that they are making ribosomes at only 5–10% the rate of WT cells, a value consistent with the results of Figure 7. The important conclusion from this experiment is that cells are able to adjust their transcription of rRNA in response to the insult of reduced production of RPs. Figure 7.Slow transcription and processing of rRNA in mutant cells. Cultures of YNN281(WT), SHY35 (ΔFHL1) and D-105 (ΔFHL1 ΔIFH1), growing in methionine drop-out medium, were pulsed with [C3H3]-methionine (Perkin-Elmer NET061-X) at 60 μCi/ml for 2.5 min (A–C) or 10 min (D, E). Cold methionine was added to 100 μg/ml and samples were taken at the indicated times. RNA was prepared and analyzed on a denaturing gel, transferred to nylon and treated with En3Hance (Perkin-Elmer) and subjected to autoradiography for 7 days at −80. (Note that the WT lanes were loaded with RNA from half as many cells as the others.) Download figure Download PowerPoint mRNAs of ribosome-deprived cells Because Fhl1p is found almost exclusively at RP genes (Lee et al, 2002) and similar results have been reported for Ifh1p (Schawalder et al, 2004), we expected to find that in ΔFHL1 strains, the level of RP mRNA would be greatly reduced compared to that derived from other genes. However, Figure 8A shows that in comparison with ACT1, RP mRNAs are reduced marginally if at all. On the other hand, using U3 snoRNA as a loading control, the levels of both RP and non-RP mRNAs appear greatly reduced in mutant cells. To investigate more thoroughly, we examined the entire spectrum of mRNAs using an Affymetrix array (Figure 8B, D and F). The results are striking! Essentially all mRNAs are reduced similarly in the mutant cells. Note that equal amounts of total RNA were used from the WT and the mutant strains to make biotin-labeled cRNA, to be hybridized to the arrays (see Materials and methods). Since the total RNA level in ΔFHL1 and ΔFHL1 ΔIFH1 cells is about a fifth of that in WT cells (Figure 5C), RNA from the mutant cultures represents five times as many cells as WT. Thus, on a per cell basis, all the genes lying on the x=0 axis in the genome-wide gene expression patterns (Figure 8D and F) are approximately five times under-represented in the mutant strains. Of the ∼5000 authentic genes analyzed, less than 300 showed signals that were preferentially increased or decreased more than two-fold in the mutant strains compared to the WT. Although there was substantial consistency between the two mutant strains, no discernable pattern appeared. For example, few of the more than 100 genes involved in ribosome biogenesis fell into this category. Figure 8.mRNA levels of ΔFHL1 and ΔFHL1, ΔIFH1 mutant strains. (A) Northern analysis showing the levels of RP mRNAs when normalized by U3 snoRNA or by ACT1. Total RNA (7.5 μg) was analyzed on denaturing agarose gels, transferred to nylon membrane and analyzed using labeled oligonucleotide probes directed against the indicated RNA species as previously described (Nierras and Warner, 1999). (B–G) Graphical representation of differential gene expression comparing DR36 (WT) with itself (B), with DR34 (ΔFHL1) (D) and with DR35 (ΔFHL1 ΔIFH1) (F). The differential expressions of only the RP genes for the above samples are shown in (C, E and G), respectively. RNA from DR36, DR34 and DR35 strains was analyzed in duplicate using individual Affymetrix S98 arrays. The robust multiarray average (RMA) algorithm was used to normalize all six arrays and to compute average gene expression values for each strain. The original data are available in Supplementary Table I. Download figure Download PowerPoint Considering that Fhl1p binds almost exclusively to RP genes, the interesting result is that we observed only marginal deficits of the RP gene transcripts (Figure 8C, E and G). Of the 115 RP genes examined, only 11 from the ΔFHL1 strain and 16 from the ΔFHL1 ΔIFH1 strain have mRNA at less than half their normal level compared to the bulk mRNA. For the most part, these were the same genes. The raw data from the array hybridization suggest that the levels of mRNA in the mutant strains are reduced to approximately the same extent as the levels of total RNA. This result demonstrates that the mutant cells have a remarkable capacity to detect a deficiency of ribosomes and to respond by reducing the amounts of all mRNAs to ensure that the mRNA/ribosome ratio is maintained within narrow limits, presumably, although not necessarily, through reduced Pol II transcription. Such ability of the translation system to provide global feedback to the transcription system, in the interests of maintaining homeostasis, is yet another example of cellular control mechanisms of which we remain profoundly ignorant. Layers of control It is evident that a certain level of transcription of RP genes can occur in the absence of the transcription factors Fhl1p and Ifh1p (Figure 8). Is this residual transcription subject to the same controls as the high levels of transcription that occur in growing WT cells? Indeed, rapamycin can repress even the residual transcription of RP genes that occurs in the absence of Fhl1p and Ifh1p (Figure 9). Although the results presented in Figures 1D and 4Csuggest that rapamycin acts by inhibiting the interaction of Ifh1p with Fhl1p, this result suggests that there is an additional layer of control of RP gene transcription beyond the interaction of Ifh1p with Fhl1p. Figure 9.Rapamycin causes repression of RP genes in cells lacking Fhl1p and Ifh1p. Strains DR34, DR47, DR48, DR49, DR65 and DR35 were treated with rapamycin (0.2 μg/ml) and harvested at indicated time points. Total RNA was isolated, and 7.5 μg of RNA was analyzed by Northern blotting as described for Figure 8. Note the consistent high levels of U3 RNA from strains deficient in functional Fhl1p. Download figure Download PowerPoint Discussion New factors in the transcription of RP genes The identification of Fhl1p and Ifh1p at the promoters of RP genes (Figure 1; Lee et al, 2002; Jorgensen et al, 2004; Schawalder et al, 2004) provides a new dimension for considering the regulation of this cohort of genes. If, as the bulk of the evidence suggests, these proteins are present exclusively at RP genes, they are likely to be key factors that recruit the transcriptional apparatus. This hypothesis is supported by the finding that when transcription of RP genes is repressed, Ifh1p is no longer found at the RP promoters. In contrast, Fhl1p, like Rap1p, seems to be present at the RP promoters even when transcription is repressed. Thus, the most economical hypothesis is that recr" @default.
• W2044346282 created "2016-06-24" @default.
• W2044346282 creator A5023370938 @default.
• W2044346282 creator A5041733646 @default.
• W2044346282 creator A5061227402 @default.
• W2044346282 date "2005-02-03" @default.
• W2044346282 modified "2023-10-15" @default.
• W2044346282 title "Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins" @default.
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