Pleiotropic Effect of Disrupting a Conserved Sequence Involved in a Long-Range Compensatory Interaction in the Drosophila Adh Gene

Corresponding author: John F. Baines, University of Munich, Luisenstrasse14, 80333 Munich, Germany., baines{at}zi.biologie.uni-muenchen.de (E-mail)

Communicating editor: S. W. SCHAEFFER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Recent advances in experimental analyses of the evolution of RNA secondary structures suggest a more complex scenario than that typically considered by Kimura's classical model of compensatory evolution. In this study, we examine one such case in more detail. Previous experimental analysis of long-range compensatory interactions between the two ends of Drosophila Adh mRNA failed to fit the classical model of compensatory evolution. To further investigate and verify long-range pairing in Drosophila Adh with respect to models of compensatory evolution and its potential functional role, we introduced site-directed mutations in the Drosophila melanogaster Adh gene. We explore two alternative hypotheses for why previous analysis of long-range compensatory interactions failed to fit the classical model. Specifically, we investigate whether the disruption of a conserved short-range pairing within Adh exon 2 has an effect on Adh expression or if there is a dual functional role of a conserved sequence in the 3'-UTR in both long-range pairing and the negative regulation of Adh expression. We find that a classical result was not observed due to the pleiotropic effect of changing a nucleotide involved in both long-range base pairing and the negative regulation of gene expression.

KIMURA's (1985) classical model of compensatory evolution and its application to nucleotide sites involved in Watson-Crick (WC) base pairing within RNA secondary structures (STEPHAN 1996 ; INNAN and STEPHAN 2001 ) typically assume a symmetrical interaction between alleles at two interacting loci. Under this scenario, with alleles A and a present at the first locus and B and b at the second, the genotypes Ab and aB are both considered deleterious, while the wild-type AB and double mutant ab are selectively neutral. In terms of RNA secondary structure, genotypes ab and AB would represent WC paired nucleotides, while Ab and aB would represent mismatches. Because of its simplicity, this model is generally used to predict the outcome of experimental tests of secondary structure (e.g., HAAS et al. 1991 ; CHEN and STEPHAN 2003 ). However, it is becoming increasingly clear that in many cases (e.g., when helices do not meet the condition such that single base changes destabilize, but do not destroy the overall structure) the classical model may be too simple (CHEN et al. 1999 ). For example, the model does not adequately explain the data of SCHAEFFER and MILLER 1993 , where the compensatory process between two divergent haplotypes in the introns of the Drosophila pseudoobscura alcohol dehydrogenase gene (Adh) likely involved significant rearrangements (insertions and deletions of bases; INNAN and STEPHAN 2001 ).

Experimental evidence also suggests that the compensatory process is more complex than previously modeled. PARSCH et al. 1997 extended the phylogenetic analysis of STEPHAN and KIRBY 1993 and predicted a long-range, tertiary contact between a region just downstream of the start codon and a conserved region of the 3'-untranslated region (UTR) in Drosophila Adh (Fig 1). Site-directed mutagenesis was used to test a long-range WC base pairing between positions 819 and 1756. A synonymous mutation in exon 2 (C to T at position 819, designated mutC819T) resulted in a significant reduction in ADH activity, while a second, compensatory mutation in the 3'-UTR (G to A at position 1756, designated mutG1756A) restored activity to that of wild-type Adh levels. However, mutG1756A alone did not significantly differ from that of wild-type levels, thus not fitting classical models of compensatory evolution where both intermediate states should be deleterious (KIMURA 1985 ; STEPHAN 1996 ; INNAN and STEPHAN 2001 ).

View larger version (15K): In this window In a new window Download PPT slide   Figure 1. Phylogenetically predicted secondary structure of wild-type exon 2 and its putative tertiary contacts with the 3'-UTR (modified from PARSCH et al. 1997 ). All positions used in mutational analysis are labeled and shown in red. Positions 819, 1756, and 1762–1769 were investigated in previous studies (PARSCH et al. 1997 , PARSCH et al. 1999 , PARSCH et al. 2000 ). Brackets connected by lines indicate predicted pairing regions.

It is well known that communication between the 5' and 3' ends of mRNA plays an important role in the initiation of translation in eukaryotes (GALLIE 1991 ; SACHS et al. 1997 ). These interactions have been well documented at the protein-protein level (TARUN and SACHS 1996 ; HENTZE 1997 ; WELLS et al. 1998 ), and it has been demonstrated that a viral transcript that lacks a 5' cap and poly(A) tail achieves 5'-3' communication and initiation of translation in a similar manner (WANG et al. 1997 ; GUO et al. 2000 ). In this case, the 5'-3' interaction occurs via direct RNA-RNA base pairing between the 5'- and 3'-UTRs (GUO et al. 2001 ). Such long-range RNA-RNA interactions have been predicted for a large number of eukaryotic transcripts (KONINGS et al. 1987 ; STEPHAN and KIRBY 1993 ; PARSCH et al. 1998 ), but the results of PARSCH et al. 1997 offer the first experimental evidence of such interactions. Thus, it is important to further investigate and verify long-range pairing in Drosophila Adh with respect to models of compensatory evolution and its potential functional role.

In this study, we experimentally investigate two alternative explanations for the results of PARSCH et al. 1997 , namely, the respective local structure/function of the nucleotides neighboring positions 819 and 1756. In the first experiment, the importance of the local secondary structure in exon 2 is investigated. Free energy minimization analysis (ZUKER 2003 ) indicates that the phylogenetically predicted RNA secondary structure of exon 2 may be displaced by a more thermodynamically stable alternate structure when position 819 is changed from C to T (PARSCH et al. 1997 ). One particular pairing region with phylogenetic support that is not present in this alternate structure is that comprising the central stem (positions 793–797/833–837). We directly test the importance of this structure by introducing two synonymous mutations that disrupt the central stem (Fig 1). Thus, if the effect seen by C819T is due to the disruption of the native exon 2 structure, these mutations should produce a similar effect.

In a second set of experiments, a conserved region of the Adh 3'-UTR encompassing position 1756 is investigated. Previous studies identified a highly conserved 8-base regulatory sequence at positions 1762–1769 (PARSCH et al. 1999 , PARSCH et al. 2000 ). Deletion of this sequence resulted in a twofold increase in ADH activity due to an underlying twofold increase in mRNA levels, suggesting a functional role in the negative regulation of mRNA (PARSCH et al. 1999 , PARSCH et al. 2000 ). It is hypothesized that the conserved portion of the 3'-UTR upstream of this 8-base sequence may play a dual role in both long-range pairing and the negative regulation of Adh expression, so that any reduction in ADH activity due to a disruption of long-range pairing may be masked by a decrease in the negative regulation of mRNA (CHEN et al. 1999 ). To investigate whether the proximity of position 1756 to the 8-bp sequence confounds the ability to measure its involvement in long-range pairing with exon 2, a series of compensatory mutations is made in a background of a deletion of positions 1762–1769. The results indicate that the local structure predicted in exon 2 has no significant effect on Adh expression, whereas the conserved region of the 3'-UTR likely plays a role in both long-range base pairing and the negative regulation of Adh mRNA levels.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Site-directed mutagenesis and plasmid construction:
All constructs were derived from an 8.6-kb SacI-ClaI fragment of the D. melanogaster Adh Wa-f allele (KREITMAN 1983 ). A pUC18 plasmid containing the 8.6-kb fragment was subjected to mutagenesis using the Quick-change mutagenesis kit (Stratagene, La Jolla, CA). Two point mutations (T to C at positions 834 and 837) were introduced simultaneously into this wild-type background to create the construct designated as E2mut. To test the long-range pairing between sites 819 and 1756, individual mutations (C to T at position 819 and G to A at position 1756) were introduced into a Wa-F allele with bases 1762–1769 deleted (3; PARSCH et al. 1999 ). These constructs were designated 3mut2 and 3mut1, respectively. A final construct (3mut3) contained both of the above mutations together in the 3 background. Desired mutations were verified by sequencing before proceeding further.

P-element-mediated germline transformation:
For each mutant construct, the respective mutant SacI-ClaI fragment was inserted into the polycloning region of the YES transformation vector (PATTON et al. 1992 ). This is a P-element vector containing the D. melanogaster yellow (y) gene as a selectable marker and suppressor of Hairy-wing binding sites flanking the target DNA, which serve as an insulator of chromosomal position effects (PATTON et al. 1992 ). Constructs were introduced into an ADH-null background by microinjection of y w; Adh^fn6; 2-3, Sb/TM6 embryos (RUBIN and SPRADLING 1982 ; SPRADLING and RUBIN 1982 ). For each mutant construct, five to six independent transformed lines were generated by microinjection. To increase the number of lines, insertions on the X chromosome were mobilized to new genomic locations by crosses utilizing the 2-3 P element as a source of transposase (ROBERTSON et al. 1988 ; PARSCH et al. 1997 ). Insertions on the third chromosome containing the source of transposase (the 2-3, Sb third chromosome) are not suitable for maintaining as transformed stocks and were thus also mobilized to new genomic locations. All lines were crossed to a y w; Adh^fn6 stock following transformation or mobilization to remove the source of transposase. Lines containing single insertions were determined by Southern blotting (PARSCH et al. 1997 ). Due to possible dosage compensation effects, only lines containing autosomal insertions were used for further analysis (LAURIE-AHLBERG and STAM 1987 ; PARSCH et al. 1997 ).

ADH assays:
ADH enzymatic activity was measured by the method of MARONI 1978 , using 2-propanol as the substrate. Assays were performed on five 6- to 8-day-old males heterozygous for the respective Adh insertion following the procedure of PARSCH et al. 1997 . The total protein of the extracts was determined by the method of LOWRY et al. 1951 , and units of activity are represented as micromoles of NAD reduced per minute per milligram of total protein. Differences in activity between Adh genotypes were tested by analysis of variance (ANOVA), using a model that accounts for position-effect variation (LAURIE-AHLBERG and STAM 1987 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of the local secondary structure of exon 2:
In our first experiment, we investigate the mechanism by which mutC819T causes a significant reduction in ADH activity. Although this mutation changes a preferred alanine codon to an unpreferred codon, it is unlikely that a single synonymous codon replacement could lead to such a large difference in gene expression (CARLINI and STEPHAN 2003 ). Thus, given that involvement in secondary structure is a more likely explanation, the next question is whether the effect of mutC819T is due to the disruption of the local structure of exon 2 or to involvement in long-range pairing with the 3'-UTR.

To test the functional significance of the local structure of exon 2, site-directed mutations were made at degenerate codon positions 834 and 837, thus disrupting the central, phylogenetically predicted pairing region 793–797/833–837 (Fig 1). This genotype, designated E2mut (T834C-T837C; Fig 2), enables an indirect test of the mechanism by which mutC819T reduces ADH activity. Our logic is as follows: Given that mutC819T creates the potential for an alternative structure in exon 2 and results in a significant reduction in ADH activity (PARSCH et al. 1997 ), loss of the hairpin structure depicted in Fig 1 may be the underlying reason for this reduction. Under this scenario, targeted disruption of this structure (see sites 834 and 837 in Fig 1) should result in a similar or more extreme phenotype (i.e., a decrease in Adh expression).

View larger version (15K): In this window In a new window Download PPT slide   Figure 2. Location of mutations along the Adh transcript. Point mutations are shown by nucleotides differing from the wild-type sequence. Deletion of bases 1762–1769 is represented by dashes. Brackets indicate phylogenetically predicted pairing regions (STEPHAN and KIRBY 1993 ; PARSCH et al. 1997 ).

Lines containing the E2mut allele were generated by P-element-mediated germline transformation and compared to lines transformed with a wild-type Wa-f control. The results indicate that E2mut lines do not have reduced ADH activity relative to wild type (Fig 3). In fact, the E2mut lines have slightly higher activity (the mean ADH activity ±SE of six E2mut vs. seven wild-type lines was 118.6 ± 6.5 units vs. 108.9 ± 4.6 units), although this difference is not significant (F = 1.99, P = 0.16). Thus, the more severe disruption of the local structure of exon 2 caused by E2mut in comparison to mutC819T does not appear to significantly affect Adh expression, leaving long-range pairing as the more likely explanation.

View larger version (13K): In this window In a new window Download PPT slide   Figure 3. Average ADH activity of wild-type and E2mut lines. Values represent the mean of seven and six transformed lines, respectively. ADH activity is given in micromoles of NAD reduced per minute per milligram of total protein (multiplied by 100). Error bars represent ±1 SE. Differences between genotypes are not statistically significant.

Analysis of long-range pairing in a deletion background:
To test the hypothesis that a classical compensatory effect was not observed between the predicted paired nucleotides 819/1756 due to the proximity of a highly conserved 8-base regulatory element in the 3'-UTR (Fig 1), a series of compensatory mutations were made in a background of a deletion of this sequence. As a control, a deletion of bases 1762–1769 was used (PARSCH et al. 1999 ) and is designated 3. The compensatory-mutant alleles, 3mut1, 3mut2, and 3mut3, contain the mutations G1756A, C819T, and C819T + G1756A, respectively (Fig 2). Thus, alleles 3 and 3mut3 allow for WC base pairing, whereas 3mut1 and 3mut2 are mismatches. The mean ADH activity ±SE of lines transformed with 3 (15 lines), 3mut1 (16 lines), 3mut2 (17 lines), and 3mut3 (12 lines) alleles was 228.3 ± 6.1, 210.8 ± 5.5, 209.3 ± 8.9, and 230.3 ± 7.1, respectively (Fig 4). Note that these activity values are approximately twofold higher than those in the first experiment, due to the absence of the 8-base negative regulatory element in the 3 background. Consistent with our hypothesis, the alleles with mutations causing mismatches (3mut1 and 3mut2) had significantly lower ADH activity in comparison to the 3 control, whereas the compensatory double mutant 3mut3 did not significantly differ from the control. Comparisons between the compensatory double mutant 3mut3 and the mismatch alleles 3mut1 and 3mut2 are also significant, although they approach significance only after Bonferroni correction (Table 1). Thus, it appears that the nucleotides within the conserved region of the 3'-UTR (1756–1769) are involved in both WC base pairing and the negative regulation of Adh mRNA, and a complete compensatory interaction may be seen only in a background in which this latter function is removed.

View larger version (17K): In this window In a new window Download PPT slide   Figure 4. Average ADH activity of 3, 3mut1, 3mut2, and 3mut3 lines. Values represent the means of 15, 16, 17, and 12 transformed lines, respectively. Units of activity are given as in Fig 3. Error bars represent ±1 SE. Tests of significance among genotypes are found in Table 1.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we examined long-range compensatory interactions between the two ends of Adh mRNA in more detail. Previous mutational analysis by PARSCH et al. 1997 suggested a long-range interaction between positions 819 and 1756, although the results did not completely conform to the classical model of compensatory evolution. We have now extended the analysis of PARSCH et al. 1997 with respect to this scenario and asked the following question: Do these results not fit the model (1) because the first deleterious intermediate (mutC819T) causes a reduction in ADH activity via a mechanism other than long-range base pairing or (2) because the deleterious status of the second intermediate (mutG1756A) is more complex due to pleiotropy?

To address the first possibility, we investigated an alternative explanation for the 15% reduction in ADH activity seen in mutC819T lines (PARSCH et al. 1997 ). More specifically, should disruption of the local structure of exon 2 be the reason for this change, disruption of this structure by two targeted mutations should produce a similar or more extreme phenotype. However, although the difference is not significant, E2mut lines have, on average, higher levels of ADH activity than do lines transformed with the wild-type Wa-f. Given that T834C and T837C each change an unpreferred codon to a preferred codon (for isoleucine and glycine, respectively), another possible explanation is that any decrease in Adh expression caused by a disruption of the structure of exon 2 may be masked by an increase in expression due to the use of preferred codons (CHEN et al. 1999 ). However, this would require the change of two synonymous codons to result in a >15% increase in expression. The recent results of CARLINI and STEPHAN 2003 suggest that such a difference may require on average seven to eight codon changes, making this scenario very unlikely. The lack of a measurable effect by disruption of the local structure of exon 2 is also in qualitative agreement with the analysis of CARLINI et al. 2001 , who show a lower potential for secondary structure in the highly expressed Adh relative to the lowly expressed Adhr gene.

In contrast, closer inspection of a conserved region in the 3'-UTR has yielded results consistent with our hypothesis concerning position 1756. Namely, mutG1756A's role as a putative deleterious intermediate may be understood only in the context of the sum of the functional roles in which it and its neighboring nucleotides are involved. Detailed information regarding an 8-base regulatory element in the 3'-UTR has proven particularly important. Previous studies have shown this sequence to be completely conserved across all Drosophila species examined (spanning the subgenera Sophophora and Drosophila, as well as the genus Scaptodrosophila) and deleting the first four bases, last four bases, or the entire sequence results in the same phenotype (a twofold increase in ADH activity due to an underlying twofold increase in mRNA; PARSCH et al. 1997 , PARSCH et al. 1999 ). Despite the apparent positive selection for increased ADH activity in the wild (i.e., the S F amino acid replacement; OAKESHOTT et al. 1982 ; BERRY and KREITMAN 1993 ; MERCOT et al. 1994 ), there appears to be strong purifying selection against changes in this sequence. Indeed, PARSCH et al. 2000 have demonstrated that transformed lines lacking this 8-base sequence (3) have a significantly delayed development time, likely due to the presence of excessive amounts of Adh mRNA.

Given our knowledge of this 8-base sequence and our results from 3mut1, 3mut2, and 3mut3 lines, we propose that mutG1756A produces a partial 3 phenotype. Under this scenario, the conserved sequences upstream of positions 1762–1769 also play a role in the negative regulation of mRNA, although to a lesser degree. One possibility is that positions 1762–1769 are essential to the binding of some trans-acting regulatory factor, whereas the conserved sequences upstream only facilitate this binding and hence produce only a partial phenotype. As demonstrated by the deletion analysis of PARSCH et al. 1999 , disruption of any part of bases 1762–1769 does produce a full phenotype, enabling position 1756's role in long-range pairing to be investigated in the absence of its role in the regulation of mRNA (i.e., there will be no pleiotropic effect of changing this nucleotide to determine its role in long-range pairing). Indeed, in a background of deleting positions 1762–1769, we show that positions 819/1756 do fit a classical model of compensatory evolution; i.e., both intermediate states show a reduction in activity (KIMURA 1985 ; STEPHAN 1996 ; INNAN and STEPHAN 2001 ).

Additional support for a dual functional role of bases 1756–1761 comes from two sources. First, phylogenetic comparisons indicate that these six nucleotides are conserved within the Sophophora subgenus, including the distantly related D. pseudoobscura and D. ambigua (PARSCH et al. 1997 ). Such strong conservation in a noncoding region suggests functional constraint. Second, the experiments of PARSCH et al. 1997 show that the single mutation G1756A results in higher ADH activity than do both the wild type and the compensatory double mutant, C819T-G1756A. Although this difference is not significant in both of the above comparisons, the qualitative pattern of ADH activity in these mutant constructs is in agreement with the above hypothesis.

	ACKNOWLEDGMENTS

We thank Jon Bollback, David Carlini, Ying Chen, and Stephan Hutter for assistance in the lab and two anonymous reviewers for helpful comments on the manuscript. This study was supported by National Institutes of Health grant GM-58404 and funds from the University of Munich to W.S.

Manuscript received August 7, 2003; Accepted for publication September 28, 2003.

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