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Nucleic Acids Research Pages 1289-1295  

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3[prime] end processing and alters selection of the poly(A) site
Introduction
Materials And Methods
   Yeast strains
   Plasmid construction and cloning
   RNA analysis
   Anthranilate phosphoribosyl transferase assay
   Reverse transcriptase (RT)-PCR
Results
   Inversion of the TRP4 3[prime] end formation element results in correct TRP4 gene expression
   The TRP4 3[prime] end formation element can be replaced by the bidirectional ARO4 or the unidirectional GCN4 3[prime] end formation element
   Deletion of the 3[prime]UTR significantly reduces TRP4 transcript level and enzyme activity
   A single point mutation within the 3[prime]UTR has the same effect on TRP4 gene expression as the deletion
   The 3[prime] end formation efficiency motif UTR-E is required for correct poly(A) site selection of the TRP4 mRNA
Discussion
Acknowledgements
References

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3[prime] end processing and alters selection of the poly(A) site

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3[prime] end processing and alters selection of the poly(A) site

Katrin Düvel, Christoph M. Egli and Gerhard H. Braus*

Institute of Microbiology and Genetics, Georg-August-University, Grisebachstraße 8, D-37077 Göttingen, Germany

Received November 23, 1998; Revised and Accepted January 15, 1999

ABSTRACT

The yeast TRP4 mRNA 3[prime] end formation element is a bidirectional element which functions in both orien-tations in an artificial in vivo test system. In this study, the role of 3[prime] end formation was analysed in the context of the entire TRP4 gene. The 3[prime] untranslated region (3[prime]UTR) of TRP4 was altered and changes were analysed for their influence on TRP4 gene expression. The 3[prime]UTR in reverse orientation was fully functional and did not affect TRP4 gene expression. Exchanging the TRP4 3[prime]UTR by the bidirectional ARO4 or the unidirectional GCN4 3[prime] end formation element allowed efficient gene expression. Deletion of the entire TRP4 3[prime]UTR resulted in 70% reduction of TRP4 mRNA and 50% reduced specific Trp4 enzyme activity in comparison to wild-type. A single point mutation within the TRP4 3[prime]UTR caused the same effect on gene expression. This point mutation did not only affect the efficiency of 3[prime] end formation, but also produced new poly(A) sites which were situated upstream of the wild-type poly(A) sites. Therefore this sequence motif in the TRP4 3[prime]UTR acts simultaneously as both an efficiency and positioning element.

INTRODUCTION

3[prime] end processing of mRNA involves two steps, namely cleavage of the pre-mRNA followed by the addition of adenosine residues at the cleavage site. The signal sequences directing this process are less conserved in yeast than in higher eukaryotes. In higher eukaryotes a highly conserved AAUAAA sequence motif upstream of the poly(A) site and a GU-rich region downstream of the poly(A) site are involved in mRNA 3[prime] end processing (reviewed in 1). In yeast, all sequence motifs required for this process are normally located upstream of the poly(A) site. Investigations of 3[prime] untranslated regions (3[prime]UTRs) of different yeast genes revealed a variety of sequence motifs (reviewed in 2). A synthetic signal sequence which was functional in yeast demonstrated the sequence requirements for efficient 3[prime] end processing of the yeast gene CYC1. This signal sequence consisted of three motifs in a defined distance to each other: (i) a far upstream motif which was essential for efficient 3[prime] end processing, (ii) a near upstream motif necessary for the positioning of the poly(A) site where cleavage occurs and (iii) the poly(A) site itself (3).

Efficiency motifs are usually located upstream of the positioning motif. An exception is the efficiency motif of the yeast FBP13[prime] end formation element which is located downstream of a poly(A) site selection motif (4). Sequence motifs which direct efficiency of 3[prime] end formation vary considerably in different yeast genes. First investigations of the CYC1 gene revealed a tripartite sequence TAG..TATGT...TTT (5,6). Variations of this motif have also been found in the TRP1, ARO4 and TRP4 genes (7). A 3[prime] end formation signal of the Cauliflower mosaic virus (CaMV) was functional in yeast because of a condensed version of the tripartite sequence motif (8). Saturation mutagenesis of the CaMV sequence revealed different functional motifs of which TATATA was the most efficient (9). In some other genes a variation of TATATA, a TTTTTAT stretch, has been found to function as an efficiency motif (10). Among others, the GCN4 and the PHO53[prime] end formation elements include such motifs (7). The GCN4 3[prime] end formation element carries two TTTTTAT stretches. Deletions of either one or both sequences resulted in 30 and 60% decreased processing activity, respectively, in an artificial in vivo test system (11). This suggests an accumulative effect of both signals in mRNA 3[prime] end formation. In most genes, 3[prime]UTRs comprise redundant degenerate efficiency motifs (12). The ADH2 and the TRP4 3[prime] end formation elements are the only described cases where a single point mutation in one of these motifs led to a total abolishment of 3[prime] end processing in a highly transcribed test gene (13,14).

A saturated mutagenesis of sequence motifs involved in the positioning of the poly(A) site of the CYC1 gene revealed AAUAA as the preferred motif (15). This sequence or variations like AAGAA or AAAAA can be found in many, but not all, yeast genes. Deletions of these motifs normally have no effect on the efficiency of 3[prime] end formation (16). An exception is the GAL7 transcript which is not processed when a crude deletion of 14 bp, including an AAUAA motif, is located in the 3[prime]UTR of the gene (17). It has been found that cleavage occurs preferentially at PyA sequences and that poly(A) sites can be scattered over a region of 200 bp. Mutagenesis of the adenosine residues resulted in alterations of poly(A) site selection (18).

Testing different 3[prime] end formation elements in an in vivo test system resulted in additional classifications (7). First, the efficiency of 3[prime] end processing varies for different 3[prime]UTRs. Weak elements were not able to process all transcripts that have been initiated at the strong ACT1 promoter used in the test system. Second, some 3[prime] end processing elements could function in both orientations (bidirectional) and others only in their natural orien-tation (unidirectional). The TRP4 and the ARO4 3[prime] end formation elements used in this study were classified as weak elements which function bidirectionally in an in vivo test system while the GCN4 element represents a strong unidirectional element (7).

Most of the information about 3[prime] end processing elements was obtained from artificial in vivo test systems. Here, we report the influence of the 3[prime] end formation element on transcription and enzyme activity of the authentic TRP4 gene. We tested the TRP4 3[prime]UTR in both orientations and exchanged it by the bidirectional ARO4 and the unidirectional GCN4 3[prime] end formation element. These constructs allowed efficient TRP4 gene expression. A single point mutation in the TRP4 3[prime]UTR resulted in the same decrease of TRP4 gene expression as a deletion of the entire TRP4 3[prime]UTR. Additionally, this point mutation resulted in alterations of poly(A) site selection. This is the first example of a single point mutation affecting both efficiency and poly(A) site positioning of mRNA 3[prime] end formation in the budding yeast Saccharomyces cerevisiae.

MATERIALS AND METHODS

Yeast strains

The yeast strain RH2063 (MATa, trp4::URA3, gcn4-101, leu2) is a derivative of the S.cerevisiae laboratory standard strain X2180-1A (MATa gal2 SUC2 mal CUP1). The TRP4 open reading frame (ORF) and the 3[prime]UTR up to the EcoRV site have been replaced by the URA3 gene. Yeast strains were cultivated on YEPD medium or on SD minimal medium with appropriate supplements at 30°C (19).

Plasmid construction and cloning

A 3.0 kb SalI fragment containing the TRP4 gene was cloned into the SalI site of pSP65 (Promega). The resulting plasmid pME982 was the basis for the construction of all TRP4 genes carrying different 3[prime] end formation elements. Deletion of the 3[prime]UTR was achieved by NdeI/EcoRV digestion, making blunt ends followed by religation. For the single point mutation the EcoRV/NdeI fragment of pME901 (14) was cloned into the NdeI/EcoRV sites of pME982. Reverse orientation of the TRP4 3[prime] end formation element was achieved by cloning the NdeI/EcoRV fragment of pME982 in reverse orientation after blunt-ending. The TRP4 3[prime]UTR was replaced by the 325 bp fragment of the ARO4 3[prime]UTR by HpaI/EcoRV restriction of pME638 (20), making blunt ends and ligation into the EcoRV/NdeI blunted sites. The 205 bp fragment of the GCN4 3[prime]UTR cloned into pME806 (11) was amplified by PCR using oligonucleotides OLCE4 (5[prime]-CGCGGGTACCCGCTGATTTCATTTACC-3[prime]) and OLCE6 (5[prime]-gcgcagatctcccccatcgtgagtg-3[prime]) (Gibco BRL), restricted with Asp18/BglII, blunt-ended and cloned into the EcoRV/NdeI blunted side. The restriction sites were provided with the primers. The orientation of these constructs was determined by PCR and restriction analysis. All constructs were cloned as 3.0 kb SmaI/PstI fragments, containing the full TRP4 gene with its different 3[prime] end formation elements, into the SmaI and PstI sites of pRS315 (21).

RNA analysis

Total RNA from S.cerevisiae was isolated according to Cross and Tinkelenberg (22). For northern analysis, 20 µg of total RNA were separated on a formaldehyde agarose gel, transferred to a positively charged nylon membrane (Biodyne B, PALL) by electroblotting and hybridised with 32P-labelled DNA fragments as previously described (11). The DNA fragments were randomly radiolabelled using the HexaLabel DNA Labelling Kit from MBI Fermentas. Band intensities were visualised by autoradiography and quantified using a BAS-1500 Phospho-imaging scanner (Fuji).

Anthranilate phosphoribosyl transferase assay

All enzyme activities were determined in situ after permeabilisation with Triton X-100 (23). Anthranilate phosphoribosyl transferase (PRtransferase, E.C. 2.4.2.18) activity was determined as described before (24) by measuring fluorometrically the decrease of anthranilate in the presence of N-(5[prime]-phosphoriosyl-1-) anthranilate isomerase. The intrinsic PRtransferase activity of the helper strain RH218 (TRP1/EcoRI circle) was always <10% of the activity of the cell extract which was assayed, and was deduced from respective values in the assays. Protein determination of whole Triton X-100-permeabilised cells was done as previously described (25).

Reverse transcriptase (RT)-PCR

Reverse transcription of 3[prime] ends of TRP4 mRNA was performed using the Superscript[trade] reverse transcriptase (Gibco BRL). 100 ng of total RNA of the different S.cerevisiae strains were reversely transcribed with a poly-18 dT primer. In a following PCR reaction, 3[prime] ends of the different TRP4 transcripts were amplified together with the TRP4-specific primer TRP-RTF (5[prime]-ATTATCTAGACCGGCAAGTACCACCTTGGC-3[prime]). DNA fragments were isolated and cloned into pBluescript SK+ (Stratagene). The poly(A) site was determined by sequencing using the T7 Sequencing Kit (Pharmacia).

RESULTS

Inversion of the TRP4 3[prime] end formation element results in correct TRP4 gene expression

The TRP4 3[prime] end formation element has been shown to function in both orientations in an in vivo test system (14). We were interested whether the inversely orientated element is also functional in the authentic TRP4 gene and how such a change would affect gene expression. For this purpose, the 147 bp fragment of the TRP4 3[prime]UTR was cloned in negative orientation. The TRP4 3[prime]UTR contains a TAGT sequence which has been shown to affect 3[prime] end processing in the in vivo test system (14). It is called UTR-E, for 3[prime]UTR-efficiency motif, and is found at position +26 relative to the translational stop codon (Fig. 1). Reverse orientation of the TRP4 3[prime]UTR changed the location of UTR-E. Formerly on the antisense strand, it was now located on the sense strand and the first T of UTR-E was situated 138 bp downstream of the translational stop codon. This construct and the others described in the following chapters were cloned into a single copy plasmid and transformed into the yeast strain RH2063 carrying a TRP4 deletion and a mutated GCN4 gene. A defective GCN4 gene prevents an interference of the transcriptional general control system of amino acid biosynthesis in case of starvation for tryptophan. Gene expression was determined by TRP4 mRNA quantification in northern experiments and measuring TRP4 encoded specific PRtransferase activity. The data obtained were compared to data from a strain harbouring the wild-type (wt) TRP4 on the same single copy plasmid.


Figure 1. Schematic presentation of the TRP4 3[prime]UTR and of constructs with different 3[prime] end formation elements which have been modified within the NdeI/EcoRV region of the TRP4 3[prime]UTR. The orientation of the 3[prime] end formation motifs is indicated by horizontal arrows and the wt major poly(A) site by a vertical arrow. Construct [Delta]3[prime]UTR is a deletion of the NdeI/EcoRV fragment of the TRP4 3[prime]UTR. The following construct dUTR-E carries a point mutation in the sequence motif UTR-E indicated by a cross (for details see text). In the third construct, the TRP4 3[prime]UTR has been cloned in negative orientation. The last two constructs represent replacements by ARO4 and GCN4 3[prime]UTRs, respectively. The entirely different GCN4 signal sequences are underlayed with light grey boxes in contrast to the dark grey boxes of the ARO4 and TRP4 motifs.

Growth tests on SD medium with and without supplementation of tryptophan showed no growth phenotype for a strain carrying TRP4 with negatively orientated 3[prime]UTR. TRP4 mRNA levels were similar to wt TRP4 levels (Fig. 2A) and the absence of additional bands implies that 3[prime] end processing did happen close to wt poly(A) sites. The TRP4 mRNA with inverted 3[prime]UTR was efficiently translated and resulted in a PRtransferase activity of 130% of wt activity (Fig. 2B). These data demonstrate that the bidirectional TRP4 3[prime] end formation element functions as efficiently in forward as in reverse orientation in the authentic TRP4 gene.


Figure 2. Gene expression of TRP4 constructs carrying different 3[prime] end formation elements. (A) Determination of mRNA level of TRP4 constructs carrying the TRP4 3[prime]UTR in negative orientation and replacements by the ARO4 and the GCN4 3[prime]UTR, respectively, compared to wt. The TRP4 transcript was ~1.2 kb in length and was visualised by autoradiography with a radiolabelled 1.5 kb MluI TRP4 DNA fragment. As constitutive control a 0.5 kb ClaI fragment of the ACT1 gene was hybridised. Quantification of transcripts by phosphoimager analysis is shown below. Strains carrying the wt TRP4 3[prime] end formation element were set as 100%. The bars represent an average of at least four independent measurements. The standard deviation did not exceed 15%. (B) Effects of modified 3[prime] end formation elements on Trp4 protein have been measured by its PRtransferase activity. In this assay the decrease of anthranilate is measured using fluorescence spectroscopy as previously described (24). One unit of specific enzyme activity is defined as 1 mM anthranilate × min-1 × mg protein-1. Specific enzyme activity of cells carrying the wt plasmid was set as 100%. This corresponded to 1.0 mM × min-1 × mg-1. The bars represent an average of five independent measurements. The standard deviation did not exceed 20%.

The TRP4 3[prime] end formation element can be replaced by the bidirectional ARO4 or the unidirectional GCN4 3[prime] end formation element

3[prime] end formation elements of yeast genes are different from each other in their sequence motifs involved in 3[prime] end processing. We were interested whether an exchange of the TRP4 3[prime]UTR by 3[prime] end formation elements of other yeast genes would affect gene expression.

The ARO4 3[prime]UTR is an example for a 3[prime] end formation element with features similar to the TRP4 element. The ARO4 3[prime]UTR functions bidirectionally in the in vivo test system and carries two putative efficiency motifs which are located 18 and 98 bp downstream of the translational stop codon. Whereas the proximate seems to be necessary for efficient processing, the more distant motif can be removed without significantly affecting 3[prime] end processing activity (26). A 327 bp spanning fragment of the ARO4 3[prime]UTR was cloned downstream of the TRP4 ORF to find out whether the ARO4 3[prime] end formation element would allow efficient TRP4 gene expression (Fig. 1). The fragment started 13 bp from the stop codon within the ARO4 ORF and included the efficiency motif and the major poly(A) site. Strains harbouring this TRP4-ARO4 hybrid revealed an amount of TRP4 mRNA which was similar to wt (Fig. 2A). Surprisingly, the PRtransferase activity was even increased to 178% of the wt activity (Fig. 2B), indicating that the ARO4 3[prime] end formation element can efficiently replace the TRP4 element with even positive effects on gene expression.

Since it was possible to replace the TRP4 3[prime] end formation element by another bidirectional element, it was of interest whether a 3[prime] end formation element with different features would also allow efficient TRP4 gene expression. The GCN4 3[prime] end formation element has been taken as an example for unidirectional elements which function only in the natural orientation in the in vivo test system. As efficiency motifs it carries two TTTTTAT sequences which are located 40 and 102 bp downstream of the translational stop codon (11). A 205 bp fragment of the GCN4 3[prime]UTR including the two TTTTTAT motifs and the poly(A) site has been cloned downstream of the TRP4 ORF (Fig. 1). The mRNA level expressed by the TRP4-GCN4 hybrid was at least as high as the wt TRP4 level (119%). A slight increase could be observed for the TRP4-GCN4-derived PRtransferase activity which was 133% of wt activity (Fig. 2A and B). Consequently, a unidirectional 3[prime] end formation element like the GCN4 element can act as an alternative for the TRP4 element despite of its different features. In summary, TRP4 gene expression does not depend on the specific TRP4 3[prime] end formation element, since replacements by a similar 3[prime] end formation element or an entirely different element did not affect gene expression in a negative way.

Deletion of the 3[prime]UTR significantly reduces TRP4 transcript level and enzyme activity

The maximum effect of TRP4 3[prime] end formation on gene expression was achieved by deleting a 147 bp fragment of the entire 3[prime]UTR containing all essential sequences for 3[prime] end formation (14) (Fig. 1). This construct is referred to as TRP4 [Delta]3[prime]UTR. Growth experiments of a strain carrying TRP4 [Delta]3[prime]UTR on SD medium lacking tryptophan revealed growth rates similar to cells carrying wt TRP4 (data not shown). The TRP4 mRNA level of the TRP4 [Delta]3[prime]UTR construct was reduced to 30% of wt level independently of a supplementation with tryptophan (Fig. 3A). Additional bands resulting from transcripts of significant difference in length could not be detected, suggesting that no stable readthrough transcripts are present in the cell. The PRtransferase activity of strains carrying TRP4 [Delta]3[prime]UTR was reduced to 58% of wt activity, implying that the TRP4 [Delta]3[prime]UTR transcripts are efficiently translated (Fig. 3B). These results show that an extensive deletion of the entire TRP4 3[prime]UTR halves gene expression, but the reduced expression does not result in a growth phenotype.


Figure 3. TRP4 gene expression of alleles carrying a deletion and a specific point mutation in the 3[prime]UTR. (A) The steady state TRP4 mRNA levels were determined by northern hybridisation using total RNA from yeast strains carrying TRP4 wt, a deletion of the TRP4 3[prime]UTR and an allele carrying a specific point mutation in the TRP4 3[prime]UTR (dUTR-E). Northern analysis was performed as described in the Materials and Methods and the legend of Figure 2A. ACT1 RNA served as constitutive control and the quantification by phosphoimager analysis is shown below. Wild-type level was set as 100%. The bars represent an average of four independent measurements and the standard deviation did not exceed 15%. (B) Effects on the Trp4p level determined by measuring PRtransferase activity. PRtransferase activity of cells harbouring the different plasmids has been determined as described in the Materials and Methods and Figure 2B. The enzyme activity of cells carrying the wt plasmid was set as 100%. The bars represent an average of five independent measurements. The standard deviation did not exceed 20%.

A single point mutation within the 3[prime]UTR has the same effect on TRP4 gene expression as the deletion

A specific point mutation in the TRP4 3[prime]UTR had been shown to abolish 3[prime] end processing activity in the in vivo test system lacking the TRP4 ORF (14). This prompted us to analyse the influence of this point mutation on gene expression of the authentic TRP4 gene. The point mutation results in an exchange from A to G at position +29 relative to the TRP4 translational stop codon, which is the last base of a TAGT motif located on the antisense strand, named UTR-E. The TRP4 3[prime]UTR was replaced by the mutated version and the resulting construct was called dUTR-E (Fig. 1). The data obtained from this construct were compared to strains carrying wt TRP4 and TRP4 [Delta]3[prime]UTR. Like the TRP4 [Delta]3[prime]UTR carrying strain, the strains harbouring the point mutation showed no growth phenotype. In northern experiments the dUTR-E-carrying strains showed TRP4 transcripts of similar sizes to the wt TRP4 strain. However, the TRP4 mRNA level was reduced to 32% of wt level which was a similar effect to in the TRP4 [Delta]3[prime]UTR strain (Fig. 3A). The same correlation can be observed for the PRtransferase activity, which is reduced in dUTR-E-carrying strains to 55% of wt activity (Fig. 3B). These data demonstrate that the UTR-E motif is crucial for mRNA 3[prime] end formation efficiency in the TRP4 3[prime]UTR of the authentic gene.

The 3[prime] end formation efficiency motif UTR-E is required for correct poly(A) site selection of the TRP4 mRNA

RT-PCR was performed to find out whether the identified efficiency motif UTR-E also affects cleavage and maturation of the different pre-mRNAs. The wt TRP4 revealed two poly(A) sites in RT-PCR experiments at positions +68 and +72 relative to the translational stop codon (Fig. 4). In case of the reverse orientation of the TRP4 3[prime]UTR, a new poly(A) site was located 28 nt downstream of the former EcoRV site (position +177 compared to the translational stop codon). The distance between the UTR-E motif and the poly(A) site in wt and reverse orientation is very similar with 43 and 39 nt, respectively (Fig. 4). In the case of the TRP4 [Delta]3[prime]UTR construct, in which all mapped wt poly(A) sites and the UTR-E motif were removed, two new poly(A) sites were located 28 and 37 nt downstream of the UAG stop codon (originally positions +166 and +172). Destruction of the UTR-E motif resulted in poly(A) sites which were located further upstream relative to the translational stop codon. The dUTR-E construct, carrying a single point mutation in UTR-E, identified poly(A) sites at positions +36, +43 and +52. These data show that a single point mutation affects efficiency and poly(A) site selection of the TRP4 mRNA 3[prime] end formation process and suggest that UTR-E is a sequence motif involved in both functions. Interestingly, the poly(A) sites of the TRP4 [Delta]3[prime]UTR and the dUTR-E construct were located in a distance of ~40-50 bp to another TAGT motif located in the ORF.

   A
   B

Figure 4. (A) Sequence data of RT-PCR. RT-PCR was performed with wt TRP4 RNA, the deletion construct [Delta]3[prime]UTR, the specific point mutation dUTR-E and the 3[prime]UTR cloned in negative orientation. Each picture represents sequence reactions of at least three independent clones. The startpoint of the poly(A) tail is indicated by an arrow. (B) Schematic presentation of poly(A) sites of constructs carrying the TRP4 3[prime]UTR cloned in negative orientation, a deletion of the TRP4 3[prime]UTR and the specific point mutation (dUTR-E) compared to wt poly(A) sites. 3[prime] end processing motifs are represented by dark grey boxes, motifs which carry point mutations (small letters) are outlined by diagonal stripes. The poly(A) sites which are represented by vertical arrows have been determined by RT-PCR and sequencing of the amplified fragments (A). The sequences have only been shown for the 3[prime] end formation motifs and the dinucleotides forming the poly(A) site. Numbering of the sites is relative to the first nucleotide following the UAG stop codon, designated as +1. The distance between TAGT motifs and the first poly(A) site of each construct includes all nucleotides of the sequence motifs.

DISCUSSION

We have shown here that a single point mutation in the 3[prime]UTR of TRP4 influences the efficiency of TRP4 gene expression on mRNA and protein levels and alters the position of poly(A) sites. In addition, we demonstrate that the TRP4 3[prime]UTR can be inverted without affecting gene expression. Other 3[prime]UTRs, such as the ARO4 3[prime]UTR or the GCN4 3[prime]UTR, are capable of replacing the corresponding TRP4 region without negative effects on TRP4 transcript or enzyme levels.

The TRP4 3[prime] end formation element has been investigated earlier in an artificial in vivo test system which consisted of the strong ACT1 promoter and the 3[prime] end formation element of the ADH1 gene. In between, different modified TRP4 3[prime]UTRs have been cloned (14). This resulted in a transcript which could be quantified but had the drawback that no protein was encoded by the transcript. The results obtained from the test system allowed us to select a mutation within the TRP4 3[prime]UTR which affected3[prime] end processing activity there. The primary aim of this work was to assay if the results with the test system are transferable to the authentic gene. In addition, we wanted to know how we could inactivate the 3[prime] end formation signal and how inactivation of the 3[prime] end formation signal affected TRP4 gene expression. The data we obtained for TRP4 gene expression verified those obtained from the test gene. Minor differences between the authentic TRP4 and the test system can be explained by the use of the strong ACT1 promoter in the test system. Differences between an efficient3[prime] end formation element like the GCN4 and a less efficient element like the TRP4 element could only be observed in the test system, where the less efficient signal could not process all transcripts initiated from the ACT1 promoter (11,14). Both classes of 3[prime] end formation elements are able to direct efficient gene expression on TRP4 transcript and enzyme level. This might be due to the fact that the TRP4 promoter is not highly efficient for transcription and it is not, therefore, necessary to supply this gene with a strong 3[prime] end formation signal.

The TRP4 3[prime] end formation element functions bidirectionally in an in vivo test system (14) and, as we could show here, also in the authentic TRP4 gene. For the authentic gene there is no difference in gene expression when the 3[prime]UTR is cloned in forward or in reverse orientation. Data obtained from the test system revealed a 20-40% reduction of the 3[prime] end processing activity when the element is cloned in negative orientation (14). This difference might also be caused by the promoter strength of the ACT1 promoter in the test system and might not play a role for less efficiently transcribed genes.

The TRP4 3[prime]UTR is not essential for viability of yeast cells under standard laboratory conditions on medium lacking tryptophan. Deletion of the TRP4 3[prime]UTR led to 30% of wt mRNA and 50% of protein level. A similar effect could be observed for the yeast ARO4 gene. The ARO4 encoded DAHP synthase activity was reduced to 41% in a strain carrying a deletion of the ARO4 3[prime]UTR (26). In the case of the CYC1 gene, a deletion within the 3[prime]UTR resulting in deficiency of 3[prime] end formation decreased the amount of mRNA and protein levels to 10% of wt levels (27). There are obviously differences in the importance of efficient 3[prime] end formation signals for different genes.

Cells carrying a deletion of the TRP4 3[prime]UTR were able to grow with a growth rate like wt cells even though inefficient 3[prime] end processing reduced Trp4p activity to 50%. It had been shown earlier that 25% of wt activity of the TRP4 encoded PRtransferase were rate limiting in tryptophan biosynthesis and resulted in a growth phenotype (23). Consequently, TRP4 gene expression produces a surplus of gene product in cells growing under standard laboratory conditions and reduction of gene expression does not necessarily lead to selective pressure.

Reduction of TRP4 gene expression was achieved by a deletion of the 3[prime]UTR. Interestingly, a single point mutation in the 3[prime]UTR affected TRP4 gene expression in the same intensity as the entire deletion. In the artificial test system this single point mutation led to a total abolishment of 3[prime] end processing activity (14). The drastic effect of a single point mutation is surprising since most yeast 3[prime] end formation elements carry redundant sequence motifs (12). The loss of 3[prime] end processing could be due to a change in the RNA secondary structure. Computer prediction of RNA secondary structures revealed alterations in case of the UTR-E point mutation (mfold server, http://mfold1.wustl.edu/mfold/rna/form1.cgi ).

There are two additional TA(T)GT motifs in the TRP4 3[prime]UTR. One of these motifs is located on the sense strand upstream of the UTR-E motif, the other downstream on the antisense strand like the UTR-E motif. These motifs had only minor effects on 3[prime] end processing in the in vivo test system (14) and point mutations within these motifs in addition to the point mutation in the efficiency motif did not further reduce TRP4 gene expression (data not shown). These findings further corroborated our earlier results obtained with the in vivo test gene construct.

Another important finding was that this single point mutation in the TRP4 3[prime]UTR did not only influence efficiency of 3[prime] end processing but also the positioning of the poly(A) site. The current model which is predominantly based on investigations of the CYC1 3[prime] end formation element proposed two different sequence motifs for these functions. The sequence motif which we found to be involved in both processes resembles the core part of a tripartite sequence motif which had been shown to act as efficiency motif in the CYC1 gene (5). Previously described positioning motifs were composed of entirely different sequences (15) which cannot be found in the TRP4 3[prime]UTR.

There is a major difference between positioning motifs previously described and the bifunctional sequence motif in the TRP4 3[prime]UTR. Point mutations in positioning motifs generally led to downstream cryptic poly(A) sites (15,16). In case of the TRP4 point mutation, new poly(A) sites are located upstream of the wt poly(A) site. In wt and in the inversely orientated TRP4 3[prime]UTR, poly(A) sites can be found in a minimal distance of 40-50 nt relative to the efficiency motif. Therefore, in both cases, the poly(A) sites are located downstream of the efficiency motif. This suggests that the efficiency motif of the bidirectional TRP4 3[prime] end formation element functions only in a downstream direction for the positioning of the poly(A) site and is therefore functionally unidirectional. Deletion of the TRP4 3[prime]UTR and a point mutation within the efficiency motif resulted in new poly(A) sites upstream of the wt sites. A similar distance of 40-50 nt between a TAGT motif located in the ORF and the cryptic new poly(A) sites can be observed. The TAGT sequence in the ORF might represent a backup system which would direct positioning of the poly(A) site when the 3[prime]UTR efficiency motif is destroyed. It is not known whether the C-terminus of the PRtransferase is necessary for enzyme activity.

Our findings suggest that intact efficiency motifs have an influence on cleavage of pre-mRNA. There are several possibilities how binding of protein factors to such an efficiency motif might influence poly(A) site selection. A bound factor could prevent cleavage factors from cleaving the RNA. Alternatively, a protein factor might bind and act as cleavage (mediating) factor itself catalysing cleavage or the recruitment of cleavage factors in a distance of 40-50 nt. The processing factor Rna15p has been discussed to bind to positioning motifs. SELEX experiments revealed a UGUGUAU2 sequence motif which was bound by Rna15p (28). Similar experiments with the human CstF-64 factor of which the RNA binding domain has high homology to Rna15p resulted in three conserved sequence motifs of at least 15 nt, which also were GU rich (29). Whether Rna15p could recognise the shorter and only slightly resembling UAGU motif in the TRP4 transcript remains speculative. However, RNA binding activity had also been shown for other components of the 3[prime] end processing machinery (30).

Yeast protein factors involved in 3[prime] end processing show significant homologies to the processing machinery of higher eukaryotes (31-34). On the other hand, the signal sequences in higher eukaryotes which direct 3[prime] end formation are highly conserved in comparison to yeast sequence motifs, and efficiency and positioning functions are strictly separated. In yeast, 3[prime] end processing does not necessarily need a separation of efficiency motifs and motifs directing the positioning of the poly(A) site as we could show in case of the TRP4 3[prime] end processing signal. Although the yeast processing machinery might be similar to higher eukaryotes, it seems to act much more flexibly in directing processing of the very different 3[prime] ends of yeast transcripts.

ACKNOWLEDGEMENTS

We thank Bernd Hoffmann, Oliver Valerius, Sabine Eckert and Dr Stefan Irniger for critical reading and helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung and the Fonds der Chemischen Industrie.

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*To whom correspondence should be addressed. Tel: +49 551 39 3771; Fax: +49 551 39 3820; Email: gbraus@gwdg.de

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