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Previous Article | Next Article 
Molecular and Cellular Biology, December 2002, p. 8415-8425, Vol. 22, No. 24
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.24.8415-8425.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Microbiology Department, Moyne Institute for Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Ireland,1 Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, and Departamento de Biotecnología, Instituto de Agroquímica y tecnología de Alimentos (IATA), M. L. Burjassot, Spain2
Received 17 July 2002/ Accepted 17 September 2002
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ABSTRACT |
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INTRODUCTION |
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The formation of metazoan histone mRNA 3' ends is controlled by a unique multicomponent process (14). Evidence from studies of sea urchin, rodent, and human cells defines at least three factors required for correct 3'-end cleavage of these mRNAs. The snRNA component of the U7 small nuclear ribonucleoprotein (snRNP) base pairs with a loosely conserved purine-rich element (referred to as the histone downstream element [HDE]), which lies approximately 10 to 15 nucleotides (nt) immediately downstream of the cleavage site (7, 29). A second factor, the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein, interacts with the conserved stem-loop at the 3' end of the mRNA via a unique RNA binding domain (4, 15, 25). This 32-kDa protein remains associated with the mature mRNA as it relocates from the nucleus to the polysomes in the cytoplasm (19). The third factor, an unidentified protein referred to as heat labile factor (HLF), was discovered through the ability of moderate temperature increases to disrupt mRNA 3'-end formation (17). Both SLBP (hairpin binding protein) and HLF levels fluctuate during the cell cycle; the SLBP accumulates in late G1 just prior to the accumulation of histone mRNAs, while HLF becomes limiting in G1 (23, 36). Although the U7 snRNP is constitutively expressed during the cell cycle, conflicting data exist regarding the cell cycle regulation of the interaction of U7 with the HDE (7, 21).
Paradoxically, unlike their metazoan counterparts, all Saccharomyces cerevisiae histone mRNAs appear to be polyadenylated (16). While the factors influencing yeast histone mRNA 3'-end cleavage have not yet been defined, it is thought that the general 3'-end processing machinery is involved. In this regard, histone pre-mRNAs can be processed in vitro by cleavage and polyadenylation extracts capable of processing general mRNAs (10). Furthermore, degenerate sequence motifs, which are similar to those of the positioning elements and efficiency elements and are located upstream of most yeast mRNAs, can be identified upstream of the 3' ends of yeast histone mRNAs. Despite the difference in the structures of the 3' ends of metazoan and yeast histone mRNAs, posttranscriptional events also contribute to the cell cycle regulation of yeast histone mRNAs (24, 37).
The role of posttranscriptional events in the cell cycle regulation of yeast histone mRNAs was demonstrated by experiments in which the 3' untranslated region (UTR) and sequences downstream of the cleavage sites of the histone gene, HTB1, were fused to a bacterial neomycin phosphotransferase (neo) open reading frame under the control of the GAL1 promoter (37). In the presence of galactose, the resultant chimeric mRNA accumulated in the S phase coincident with the endogenous HTB1 mRNA. Deletion of sequences in the HTB1 3' UTR abolished the cell cycle regulation of the neo-HTB1 transcript. Since the chimera gene also contained the HTB1 3'-end cleavage sites and sequences downstream, it is not clear whether additional events such as 3'-end processing or transcription termination also contribute to cell cycle regulation.
The formation of nonhistone and histone mRNA 3' ends has been shown to be tightly coupled to transcription termination (13, 27, 30, 31). In S. cerevisiae, mutations in signals controlling 3'-end cleavage and polyadenylation can reduce transcription termination efficiency (5). Additionally, mutations in a number of the trans-acting cleavage and polyadenylation factors, including Pcf11p and Rna15p, result in the disruption of both 3'-end processing and transcription termination (5). Coupling between cleavage and termination appears to be mediated through the C-terminal domain of RNA polymerase II (3, 26). Studies of mice have also revealed a link between mRNA 3'-end processing and transcription termination of histone genes. Removal of the 3'-end processing signals (consisting of the stem-loop structure at the 3' end of the RNA and the U7 snRNA binding site) of the mouse H2A-614 gene leads to a disruption of transcription termination (12). Interestingly, a physical and genetic linkage between the cleavage and polyadenylation factor CstF-64 (Rna15p) and the transcription factor Res2p, a factor involved in cell cycle regulation of a number of genes, has been demonstrated (2).
In this study, we examined the role of downstream sequences in the 3'-end processing and the cell cycle regulation of the yeast histone HTB1 mRNA. Using a neo-HTB1 chimera gene as a model system, we carried out a mutagenesis study of a region of DNA lying approximately 110 nt downstream of the cleavage sites of the HTB1 gene. We identify a sequence element that lies in the region of transcription termination which influences, in vivo, both mRNA 3'-end cleavage site selection and cell cycle regulation of the upstream mRNA. We show that RNA transcribed from this region binds a protein factor(s) and that maximum binding is observed during the S phase of the cell cycle.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation and analysis of RNA. Total RNA was isolated by the hot-phenol method (9). RNA (30 µg per lane) was separated on 1.0% formaldehyde agarose gels and subsequently blotted to Nytran membranes (Schleicher & Schuell). Digoxygenin-UTP (Roche Biochemicals)-labeled DNA probes were generated by PCR amplification using the primer pairs listed in Table 1, as previously described (9). The membranes were prehybridized for 1 to 3 h in high sodium dodecyl sulfate (SDS) buffer (7% sodium dodecyl sulfate [SDS], 50% formamide, 5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% blocking reagent, 50 mM sodium phosphate [pH 7.0], 0.1% N-lauroylsarcosine) at 50°C and hybridized for 15 to 20 h at 50°C, with approximately 25 ng of labeled probe per milliliter of hybridization buffer. Hybridization was detected with a chemiluminescent substrate, CSPD or CDP-Star (both from Roche), as previously described (9).
S1 nuclease analysis.
Two probes were used to map the 3' ends of the neo-HTB1 mRNAs. A 591-nt BamHI-StyI fragment was isolated from the plasmid pLJ31-HTB1 (Fig. 1A). A second 458-nt StyI-ScaI DNA fragment (see Fig. 3B) was used to determine the 3' end closer to the neo sequences. These DNA fragments were 3' end labeled at the BamHI and StyI sites, respectively, by using a Klenow fragment of DNA I polymerase and incorporating [
-32P]dATP (specific activity, 3,000 Ci/mmol).
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TRO assays.
Transcription run-on (TRO) assays were carried out as previously described (1), with the following modifications: yeast cultures (100 ml each), transformed with the plasmid pLJ31, were grown to an OD600 of approximately 0.1 to 0.15. Cells were harvested and washed with ice-cold distilled water (dH2O). Cells were rendered more permeable by resuspension in 0.95 ml of ice-cold dH2O and 50 µl of 10% (wt/vol) sodium N-lauryl sarcosine sulfate. The mix was incubated on ice for 20 min and then centrifuged for 1 min. The pellet was resuspended in 100 µl of transcription buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl2, 100 mM NaCl, 1 mM MnCl2, 2 mM dithiothreitol, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 100 µCi [-32P]UTP [3,000 Ci/mmol]). Transcription was allowed to proceed for 5 min at 30°C, and the reaction was stopped by the addition of 1 ml of ice-cold H2O. Pelleted cells were resuspended in 500 µl of TES buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.1% [wt/vol] SDS), and total RNA was extracted as described previously. RNA was partially hydrolyzed for 5 min on ice (0.2 M NaOH), neutralized (0.2 M Tris-HCl [pH 7.2]), and used for hybridization to DNA fragments corresponding to the untranslated and downstream regions of the HTB1 gene.
To generate HTB1 DNA fragments, DNA in 200-bp increments corresponding to the regions downstream of the neo open reading frame was amplified by PCR using oligonucleotides which include recognition sites for EcoRI (upstream oligonucleotide) or KpnI (downstream oligonucleotide) (Table 1). The amplified DNA was cloned into the M13 phage. Single-stranded M13 DNA corresponding to each 200-bp fragment was immobilized onto nylon filters. Filters were prehybridized for 2 h in a solution containing 5x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 10x Denhardt solution, 50% formamide, and 0.2% SDS. Hybridization was carried out in the same solution overnight. Filters were washed four times with 2x SSC-0.1% SDS at room temperature and 0.2x SSC-0.1% SDS at 37 to 42°C for 10 min. Run-on signals were then visualized with an Instant Imager (Packard) or an Image Reader FLA 3000 (Fujifilm).
Synchronization of yeast cells.
Strain S150-2B MATa cells containing either the pLJ31-HTB1 plasmid or its mutant derivatives were grown in selective medium (synthetic complete medium without uracil) to early log phase and were then grown overnight in YEPG medium. 1-mating factor (TRP-HIS-TRP-LEU-GLN-LEU-LYS-PRO-GLY-GLN-PRO-MET-TYR) was added to the yeast culture at a concentration of 2 µg/ml. The culture was then incubated for another 3 h at 30°C until the cells were arrested in the G1 phase of the cell cycle. Arrested cells showing the characteristic peanut shape (shmooed) appearance were identified by microscopic examination. When at least 90% of the cells had shmooed, the cells were centrifuged at 3,000 x g for 5 min at 4°C. The -factor was removed by washing the cells twice with 150 ml of ice-cold sterile distilled water, followed by centrifugation at the same speed as before. After the last wash, the pelleted cells were resuspended in 300 ml of prewarmed YEPG medium. Samples (15 ml) were then taken at 5-min intervals, spun at 3,000 x g for 5 min, and frozen at -70°C.
In vitro transcription of RNA.
RNA transcripts were generated by T7 RNA polymerase runoff transcription as previously described (8). The sequences to be transcribed were cloned downstream of the T7 promoter. Runoff transcription products were obtained by linearizing the plasmid with a restriction enzyme downstream of the sequence to be transcribed. Alternatively, templates were obtained by amplification of a DNA fragment by using a primer containing the T7 promoter fused to sequences complementary to the 5' start of the RNA transcript and a reverse primer complementary to the end of the transcript. Where radiolabeled in vitro-transcribed RNA was required, a molar ratio of 26:1 of cold rUTP-[-32P]UTP (3,000 Ci/mmol) was added.
Preparation of yeast cell extracts and protein-RNA binding assays.
Cell pellets from either synchronous or asynchronous cultures were prepared as previously described (9). Various concentrations of total protein extract were added to 5 x 105 cpm of [-32P]UTP-labeled RNA probe. Then, 1x binding buffer [10 mM Tris-HCl (pH 7.5), 5 mM Mg(OAc)2, 100 mM KOAc, 2 mM dithiothreitol, 0.1 mM spermine, 100 ng of bovine serum albumin/µl, 8U of RNasin, 0.2 µg of tRNA/µl, 10% (vol/vol) glycerol, 5 mg of heparin/ml] was added to the protein-probe mix and the volume was adjusted to 20 µl with double-distilled water (ddH2O). The reaction mixture was incubated on ice for 30 min and then loaded onto 4% (40:1 acrylamide-bisacrylamide) nondenaturing gel containing 1x Tris-borate-EDTA buffer and 5% (vol/vol) glycerol. After electrophoresis, the gel was dried and exposed to X-ray film.
Quantification of mRNA transcripts. The level of neo-HTB1 mRNA transcripts during the cell cycle was quantified by using the densitometric program Gel Works 1D Advanced, version 3.01 (Nonlinear Dynamics Ltd.).
neo-HTB1 mRNA transcripts levels were normalized with respect to the level of actin transcripts in each lane. For each data set, the value at every time point was accessed relative to the value at 20 h after the end of log-phase growth (T20) and the results were plotted accordingly.
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RESULTS |
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To ensure that the chimera neo-HTB1 mRNA was correctly processed, the 3' end of the mRNA was mapped by using S1 nuclease protection experiments (Fig. 1A). Four cleavage sites were identified and mapped to positions +104, +119, +126, and +138 relative to the position of the stop codon of the HTB1 gene (Fig. 1B). These cleavage sites correspond closely to those mapped in the endogenous HTB1 gene (data not shown and Fig. 1B), indicating that the chimera gene is authentically processed at the correct cleavage sites.
To identify potentially conserved sequences required for 3'-end processing and/or cell cycle regulation, we initially carried out a bioinformatic analysis of the sequences in the region encompassing the 3' ends of yeast histone mRNAs. The 3' ends of five of the eight yeast histone mRNAs have been mapped (20, 32, 35). These histone mRNAs were aligned with respect to their 3'-end cleavage sites by using the ClustalW multiple sequence alignment program. Using parameters that identify the features which are most conserved between members of a set of sequences, we observed that each of the histone genes contained stretches of purine-rich sequences downstream of the 3'-end cleavage sites (Fig. 1B and data not shown). Based on their locations, these purine stretches can be defined as being proximal or distal to the 3'-end cleavage sites. While not all of the histone genes contained identifiable proximal purine-rich sequences, they all contained one or more purine-rich stretches in the region of nt +60 to +120 downstream of their 3' ends (data not shown). The HTB1 gene contains three such purine rich clusters at positions +21, +73, and +106 relative to the position of the strongest cleavage site (Fig. 1B). The presence of purine-rich stretches, or "words," downstream of yeast genes is quite unusual. Two recent bioinformatics studies have shown that the most common words downstream of yeast genes are U-rich stretches, while the least common words are GA-rich stretches (18, 34). Given the known role of downstream purine-rich sequences in metazoan histone mRNA processing and the unusual distribution of GA stretches downstream of the yeast histone genes, we first focused our analysis on one of the HTB1 distal purine-rich sequences.
Mutations in the downstream sequences of the HTB1 mRNA alter 3'-end cleavage site selection and the steady-state levels of the neo-HTB1 transcript. The distal purine-rich sequence of the HTB1 gene that we have examined lies approximately 110 nt downstream of the last cleavage site of the mRNA and contains the sequence AGAAAAG (Fig. 1B). To examine the role of this sequence in the 3'-end processing and cell cycle regulation of the HTB1 gene, mutants were prepared in which purines were replaced by pyrimidines. Since the purine-rich HDE of metazoan histone mRNAs is always found in the context of the stem-loop of the histone mRNA, we also prepared a mutant (pSAC17) in which a stem-loop structure was inserted upstream of the purine-rich sequence. Additional mutations were also prepared in the sequences surrounding the purine stretch (Fig. 2A). Following transformation of the mutated plasmids into yeast cells, Northern blotting was performed on RNA extracted from cultures grown in galactose-containing medium. The blots were hybridized with a probe containing neo sequences only (Fig. 2A). As shown in Fig. 2B, a number of alterations are apparent in the neo-HTB1 transcript.
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Experiments with the mutants pSAC10, -11, and -16 resulted in the generation of a smaller transcript (transcript B; Fig. 2B, lanes 3, 4, and 5). The full-length neo-HTB1 transcript is also altered in size in these mutants (transcript A). S1 mapping showed that transcript A contained both neo and HTB1 sequences and had the same 3'-end cleavage sites as the neo-HTB1 wild-type (WT) transcript (see below).
All of the other mutants showed the same transcript pattern as cells harboring the WT neo-HTB1 transcript (Fig. 2B, lanes 6 to 10). We did observe some fluctuations in the steady-state levels of the neo-HTB1 transcript in some of the mutants (mutants pSAC10 and -20; Fig. 2B, lanes 3 and 9). In most cases this fluctuation correlated with the OD600 at which the cells were harvested, since increased transcript levels were observed as the OD600 of the culture increased. However, we consistently observed that the steady-state levels of the neo-HTB1 transcript in mutant pSAC20 were significantly lower than those of the WT transcript (Fig. 2B, lane 9). In three independent experiments in which RNA from cultures harvested at the same OD600 was used, the steady-state levels of the neo-HTB1 transcript in mutant pSAC20 were on average fivefold lower than those of the WT transcript. This mutant contains two sets of mutated triplets (Fig. 2A), including the triplet mutated in pSAC14. Since the steady-state level of neo-HTB1 mRNA in mutant pSAC14 appears normal (Fig. 2B, lane 8), we can deduce that the additional bases mutated in pSAC20 are responsible for the decrease in steady-state levels. Surprisingly, the levels of the neo-HTB1 transcript in mutant pSAC21 are similar to the WT levels (Fig. 2B, lane 10). This mutant contains the same mutated bases as mutant pSAC20 plus an additional triplet base change, suggesting that the additional changes in sequence had compensated for the decrease in transcript levels observed with the pSAC20 mutant.
Transcript B is generated by the use of an alternative 3'-end cleavage site. To determine whether the shorter transcripts (transcripts A and B) observed with mutants pSAC10, -11, and -16 resulted from the use of alternative 3'-end cleavage sites, S1 mapping using probes encompassing both neo and HTB1 sequences was performed. As shown in Fig. 3A, transcript A results from cleavage at the normal 3' cleavage sites downstream of the HTB1 sequences (Fig. 3A, lanes 3 and 4, and data not shown). Since no other 3'-end cleavage sites were identified, the size difference between the full-length transcripts generated by the WT and mutant genes may result from the presence of a shorter poly(A) tail or from transcription from an alternative initiation site. The full-length WT neo-HTB1 mRNA and transcripts A and B are all equally retained on oligo(dT) cellulose columns (data not shown). As with mutants pSAC10, -11, and -16, the full-length neo-HTB1 transcripts observed in all other mutants are cleaved at the normal 3'-end cleavage sites (Fig. 3A).
To identify the 3' ends of transcript B, S1 nuclease mapping was carried out by using a DNA probe encompassing the 3' end of the neo gene and the 5' end of the HTB1 sequences (Fig. 3B). We observed that all three mutants (pSAC10, -11, and -16) contain a new 3'-end cleavage site which lies approximately 45 nt downstream of the neo stop codon and 12 nt downstream of the BamHI site (Fig. 3B, lanes 3, 4, and 5, and C). Therefore, transcript B does not contain any HTB1 sequences. This new cleavage site was not observed in the WT mRNA or with any of the other mutants (Fig. 3B, lanes 1 and 2, and data not shown).
Mutations downstream of the HTB1 gene lie in a region of RNA polymerase II termination.
The area where the mutations were made (hereafter referred to as the distal downstream element [DDE]) lies quite distal to the 3'-end cleavage sites, and yet these mutants affect both the steady-state levels and 3'-end cleavage site selection of the neo-HTB1 mRNA. We therefore wanted to test whether the region in which the mutations were made was in fact transcribed as part of the primary pre-mRNA transcript. TRO experiments were carried out as previously described (1). This assay quantifies the density of RNA polymerase molecules along a transcript by incorporating [-32P]UTP into the elongating RNA transcript and is most effective for analysis of highly transcribed genes. Single-stranded DNA probes, corresponding to sequences located in approximately 200-nt increments downstream of the HTB1 stop codon, were prepared by the cloning of these fragments into the replicative form of the phage M13. Single-stranded DNA from the phage was immobilized onto nylon membranes and hybridized to 32P-labeled nascent RNA isolated from cells transformed with the WT neo-HTB1 plasmid (Fig. 4). To distinguish between transcripts emanating from the endogenous gene and the neo-HTB1 gene, we compared the levels of nascent transcripts in cells grown in glucose and galactose. The first DNA fragment (probe 1 [nt +9 to +196]; Fig. 1B) contains the entire 3' UTR and the 3'-end cleavage sites (nt +104, +119, +126, and +138), while probe 2 (+235 to +409) contains the DDE, which lies close to the 5' end of the fragment (nt +250; Fig. 1B). As shown in Fig. 4B, we observed transcription proceeding from the position of probe 1 to probe 2. Taking the transcription level to be 100% for probe 1 and having subtracted the background hybridization, in our analysis of galactose-containing medium we observed that transcription decreased to 63% in the probe 2 region, 24% in the probe 3 region, and 0% in the probe 4 region. Similarly, in glucose-containing medium, there was a decrease in transcription to 51% in the probe 2 region, 16% in the probe 3 region, and 0% in the probe 4 region. These results suggest that transcription termination was occurring somewhere in the region of probes 2 and 3. Analysis of run-on transcription in cells transformed with the mutated plasmids indicated that all mutant transcripts were transcribed as far as the region of probe 2, and there appeared to be no significant quantitative differences in the levels of transcription of the mutant mRNAs (data not shown).
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1-mating factor. Once the cells were synchronized, the -factor was removed and RNA was prepared from samples taken at 5-min intervals. The RNA was hybridized with a DNA probe specific to the neo region of the gene (Fig. 2A). The pattern of the endogenous histone mRNA was monitored by hybridizing the RNA samples with a probe specific to the coding region of HTB1 (endogenous HTB1 primers; Table 1). To allow for adjustment for the amount of RNA loaded in each lane, the blots were also hybridized to an actin DNA probe. As shown in Fig. 5A, the endogenous HTB1 mRNA was detected 20 min after -factor removal and peaked at 35 to 40 min and levels of the mRNA decreased at 45 to 50 min, with little or none detected at 55 min. The WT neo-HTB1 transcript (Fig. 5B) shows an accumulation pattern similar to that of the endogenous HTB1; however, the rate of decline in mRNA levels appears slower than that of the endogenous HTB1. These results confirm that the sequences in the 3' UTR and downstream of the cleavage sites can confer cell cycle periodicity on a neo mRNA (37).
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Sequences in the DDE bind protein factor(s).
The results obtained from the analysis of mutations in the DDE suggest that these sequences influence the accumulation and subsequent turnover of HTB1 mRNA during the cell cycle. Since two of the mutants showed a delay in the accumulation of and a lack of turnover of neo-HTB1 mRNA, we reasoned that a protein factor might be required to stabilize the pre-mRNA in the S phase or destabilize it immediately thereafter. To examine whether this region was capable of binding protein factors, a 100-nt [32P]dUTP-labeled RNA probe, consisting of the DDE and 80 nt of surrounding sequences (Fig. 6A), was generated by using T7 RNA polymerase. The RNA was incubated with cell extracts prepared from yeast cells harvested at different time points following release of cells from -factor synchronization. In the presence of cell extracts, the labeled RNA bound to one or more protein factors, as indicated by the retardation of the mobility of the RNA on nondenaturing gels (Fig. 6B). Interestingly, maximum binding was observed in extracts prepared at 35 min following -factor release (Fig. 6B, lanes 5 and 6), the time point corresponding to the peak of neo-HTB1 mRNA during the cell cycle. Binding is also observed at 30 min (Fig. 6B, lanes 2 to 4) and 40 min (lanes 7 to 9), with a marked decrease in binding occurring at 45 min (lanes 10 to 12).
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DISCUSSION |
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We observed that experimentation using one of the mutants, pSAC20, resulted in a fivefold or greater decrease in the steady-state levels of the neo-HTB1 mRNA. Since the mutated sequences are not contained in the final transcript, this reduction in steady-state levels of neo-HTB1 may be a result of a decrease in efficient 3'-end processing or may affect the stability of the final transcript. Interestingly, Kim et al. (22) have identified a 54-nt element downstream of the distal poly(A) site of the Schizosaccharomyces pombe uv15+ gene that can influence the stability of the mature mRNA. Like the DDE described here, the uv15+ element is not included in the mature mRNA. Analysis of the mutant pSAC20 RNA during the cell cycle revealed that the RNA, while induced in the S phase, appears to be more rapidly degraded as cells enter the G2 phase (Fig. 5D). Thus, the inherent instability of the mutant pSAC20 RNA may be a consequence of its rapid turnover in the G2 phase of the cell cycle. Taken together, the data suggest that sequences downstream of the HTB1 gene, though not part of the mature mRNA, can affect the steady-state levels of the final transcript.
It is surprising that mutant pSAC21 mRNA, which contains the same mutated bases as mutant pSAC20 (Fig. 2A) plus an additional triplet base pair change, shows steady-state transcription levels similar to those of the WT mRNA. Furthermore, mutant pSAC14 shares three of the six base pair changes of pSAC20 and it too shows normal steady-state levels of neo-HTB1 transcript. These findings suggest that the presence of a specific sequence is not alone sufficient to determine the overall function of this region and that a secondary structure may also play a role (see below).
Of the other mutants generated, pSAC13 and -17 showed normal steady-state levels (Fig. 2A). Mutant pSAC13 (AAA to TTT) includes three of the bases changed in mutant pSAC11 (AGAAA to TCTCT) (Fig. 2A). Unlike pSAC11, this mutant does not result in the generation of transcript B. Therefore, we can conclude that transcript B is generated as a result of the introduction of bases (TCT) that are common between mutants pSAC10 and -11 or, alternatively, that it is the introduction of C residues that accounts for the generation of transcript B. The latter argument is supported by findings for mutant pSAC16 in which a single base change from A to C resulted in the generation of transcript B (Fig. 2A). Finally, it is surprising that the introduction of a stem-loop structure (pSAC17) at the same location as mutant pSAC16 caused the transcript pattern to revert to that of the WT. This may indicate that the overall sequence context in this region is important to its function.
Sequences located at a distance from the 3'-end cleavage sites are known to influence transcription termination. To determine whether the DDE was transcribed as part of the pre-mRNA, TRO experiments were carried out. While we have not defined the exact position of termination, the results of this study show that transcription does indeed proceed as far as the DDE and that the majority of transcripts terminate no more than 150 nt downstream of the element.
The processes of 3'-end cleavage and polyadenylation and transcription termination are intimately linked (5). A bipartite signal consisting of a functional polyadenylation element and a downstream transcription pause element are required for efficient transcription termination (30). Mutations in the former element have been shown to reduce the efficiency of termination. Among the components of cleavage factor 1A in yeast, the most notable proteins required for efficient termination are Rna15, Rna14, and Pcf11 (30). These proteins appear to influence termination through their interaction with the C-terminal domain of RNA polymerase II (2, 11). A connection between transcription termination and 3'-end processing of the nonpolyadenylated mouse histone H2A gene has also been demonstrated (12). Removal of cis-acting sequences that are required for 3'-end processing of this gene disrupts transcription termination. The DDE described here is located just upstream of the region of transcription termination. However, according to the findings for the mutations examined in this study, it appears that these cis-acting sequences are not directly required for transcription termination (data not shown). Given the location of the element and the known connection between transcription termination and 3'-end cleavage, further mutations may uncover a link between these two processes.
Cell cycle regulation is altered by mutations in the DDE. Our data also reveal that two of the mutants in the DDE, pSAC14 and pSAC21 (Fig. 5C and E), can alter the periodic accumulation of neo-HTB1 mRNA during the cell cycle. Interestingly, these mutations, while in the same general location as those altering 3'-end cleavage, are in fact distinct. Mutations altering 3'-end cleavage do not alter cell cycle regulation, and mutations altering cell cycle regulation do not alter 3'-end cleavage. The mutations in pSAC-14 and -21 lie adjacent to one another (Fig. 2A). Using the RNA folding program MFOLD (http://www.bioinfo.rpi.edu/), we found that the sequences surrounding the DDE can be folded into a putative stem-loop structure (Fig. 7). Both mutant pSAC14 and mutant pSAC21 lie at the top end of the stem structure, while the mutants that affect 3'-end cleavage lie further down the stem. Using a band shift assay, we showed that an RNA molecule encompassing this stem-loop structure binds a protein factor or factors that are maximally present in the S phase of the cell cycle. The fact that maximum binding is observed in the S phase of the cell cycle may indicate the binding of a cell cycle-regulated factor. Both pSAC14 and -21 mutants showed a reduction and an absence of protein binding, respectively, and also showed a lack of turnover of the mRNA following the S phase, suggesting that the protein factor is required for destabilization of the mRNA following the S phase. Furthermore, mutant pSAC20, which exhibited more rapid turnover of the RNA, showed a greater degree of binding to proteins present in extracts from the S phase of the cell cycle. Using the MFOLD program, we observed that the putative secondary structure of these RNAs, in the region of the mutations, was altered (data not shown), strengthening the argument that a specific factor is interacting with this region. The sequences in the DDE that regulate 3'-end processing and cell cycle periodicity appear to act independently of each other; however, since they are so closely linked spatially, it is possible that further mutations may reveal an overlapping link between 3'-end processing and cell cycle regulation.
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ACKNOWLEDGMENTS |
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This work was supported by a grant to U.B. from Enterprise Ireland (SC/97/326) and as part of the National Development Plan (grant SC/01/398) and was partly funded by the European Community-European Regional Development Plan. M. del Olmo is funded by Generalitat Valenciana (GV99-105-1-13) and the Ministerio de Educación y Ciencia (CICYT ALI99-1224-C02-02).
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FOOTNOTES |
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X174 DNA ladder. All mutants were grown in galactose. The S1 cleavage pattern was observed in at least two independent experiments. (C) Sequences surrounding the new cleavage sites in mutants pSAC10, -11, and -16 are shown. The schematic representation of the neo-HTB1 gene is shown as outlined in the upper half of panel B. The single vertical arrow shows the location of the new cleavage site immediately downstream of the BamHI site. The sequence in this region is shown in expanded form below the schematic representation. The BamHI site is represented by underlined bold letters. The final nucleotide represents the cleavage site.
