Site-directed mutations reveal long-range compensatory interactions in the Adh gene of Drosophila melanogaster

Abstract

Long-range interactions between the 5' and 3' ends of mRNA molecules have been suggested to play a role in the initiation of translation and the regulation of gene expression. To identify such interactions and to study their molecular evolution, we used phylogenetic analysis to generate a model of mRNA higher-order structure in the Adh transcript of Drosophila melanogaster. This model predicts long-range, tertiary contacts between a region of the protein-encoding sequence just downstream of the start codon and a conserved sequence in the 3' untranslated region (UTR). To further examine the proposed structure, site-directed mutations were generated in vitro in a cloned D. melanogaster Adh gene, and the mutant constructs were introduced into the Drosophila germ line through P-element mediated transformation. Transformants were spectrophotometrically assayed for alcohol dehydrogenase activity. Our results indicate that transformants containing a silent mutation near the start of the protein-encoding sequence show an approximately 15% reduction in alcohol dehydrogenase activity relative to wild-type transformants. This activity can be restored to wild-type levels by a second, compensatory mutation in the 3' UTR. These observations are consistent with a higher-order structure model that includes long-range interactions between the 5' and 3' ends of the Adh mRNA. However, our results do not fit the classical compensatory substitution model because the second mutation by itself (in the 3' UTR) did not show a measurable reduction in gene expression.

Figure 1

Restriction map of the 8.6-kb Adh fragment used for transformation experiments. The mRNA-encoding region is shown as a box, with the solid portions representing the protein-encoding regions. An enlargement of the transcriptional unit is shown above, with exons represented as boxes and introns as solid lines. The locations of the adult and larval 5′ leader sequences are indicated. The 3′ UTR and the portion of the 5′ UTR shared by both adult and larval transcripts are shown as shaded boxes. The Adh probe used to identify single-insert lines is shown underneath as a hatched box.

Figure 2

Sequence alignment of the protein-encoding region of exon 2 and the conserved portion of the 3′ UTR of Adh alleles from 10 different Drosophila species. mel, D. melanogaster; tei, D. teissieri; ere, D. erecta; psu, D. pseudoobscura; amb, D. ambigua; hyd, D. hydei; mul, D. mulleri; aff, D. affinidisjuncta; sil, D. silvestris; leb, D. lebanonensis. The two medfly sequences are shown at the bottom (med1 and med2). The D. melanogaster sequence is that of the Wa-F allele used in our experiments. Numbering is from Kreitman (17). Gaps in the alignment are indicated by dashes. Regions that showed too much divergence for alignment are indicated by dots. The brackets at the bottom indicate the phylogenetically predicted pairing regions. Covarying sites are underlined. Note that some of the pairings are not conserved in the medfly (see text).

Figure 3

Model of the RNA structure for portions of the Adh transcript. (A) Secondary structure proposed for exon 2. Pairing regions are shown connected by straight lines. All pairing stems were determined by phylogenetic analysis (see Table 1). Note that this figure shows only the portion of exon 2 where the four conserved pairings were found. (B) Potential tertiary contacts between the conserved portion of the 3′ UTR and a region of exon 2. Positions 819 and 1756, which showed a covariation in our alignment and were the targets of site-directed mutagenesis, are indicated.

Figure 4

ADH activity of all lines (Upper) and autosomal insert lines (Lower) for the different transformant classes. Activity is given in units of μmol of NAD reduced per minute per milligram of total protein (multiplied by 100). Error bars represent the least significant difference at the 5% level. Least significant differences were calculated for individual comparisons, with mutC819T being compared with wild type, and all other lines being compared with mutC819T.