Compensatory evolution of a precursor messenger RNA secondary structure in the Drosophila melanogaster Adh gene

Abstract

Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (Adh) in diverse Drosophila species. In this study, the putative hairpin structure was evaluated systematically in Drosophila melanogaster by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in Drosophila melanogaster Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed.

Fig. 1.

Mutational analysis of the putative hairpin structure in D. melanogaster Adh intron 1. (A) Mutant constructs used in the experiments. The conserved nucleotide positions 899–904 (L) are shown in capital letters and 911–916 (R) in lowercase letters. A dash between two nucleotides indicates Watson–Crick pairing. (B) RNA secondary structure of D. melanogaster Adh intron 1 predicted by mfold 3.1 (18). The conserved pairing region is in bold, and the branch point sequence (UUAA) is shown in shadow. (C) Means and standard errors of the RSE of the WT and each mutant construct. The constructs for which the conserved secondary structure is disrupted are shown as white columns, whereas the constructs with the secondary structure restored or intact are shown in black. Confidence intervals (95%) are indicated by error bars on each column. (D) Means and standard errors of the ADH enzyme activities of the WT and the mutant transformed lines. The color scheme follows C. Confidence intervals (95%) are indicated by error bars on each column.

Fig. 2.

Mutational analysis of the secondary structure stability. (A) Mutant constructs used in the experiments. The conserved pairing regions are in bold, and the additional pairings in the mutant constructs are shown in shadow. Below each structure is the folding free energy ΔG given by mfold 3.1 (18). (B) Means and standard errors of the RSE of the WT (black) and the 9-bp stem mutant constructs (white). Confidence intervals (95%) are indicated by error bars on each column. (C) Means and standard errors of the ADH enzyme activities of the WT (black) and Mut9bp-2 (white) transformed lines. Confidence intervals (95%) are indicated by error bars on each column.