Neutral evolution of robustness in Drosophila microRNA precursors

Abstract

Mutational robustness describes the extent to which a phenotype remains unchanged in the face of mutations. Theory predicts that the strength of direct selection for mutational robustness is at most the magnitude of the rate of deleterious mutation. As far as nucleic acid sequences are concerned, only long sequences in organisms with high deleterious mutation rates and large population sizes are expected to evolve mutational robustness. Surprisingly, recent studies have concluded that molecules that meet none of these conditions--the microRNA precursors (pre-miRNAs) of multicellular eukaryotes--show signs of selection for mutational and/or environmental robustness. To resolve the apparent disagreement between theory and these studies, we have reconstructed the evolutionary history of Drosophila pre-miRNAs and compared the robustness of each sequence to that of its reconstructed ancestor. In addition, we "replayed the tape" of pre-miRNA evolution via simulation under different evolutionary assumptions and compared these alternative histories with the actual one. We found that Drosophila pre-miRNAs have evolved under strong purifying selection against changes in secondary structure. Contrary to earlier claims, there is no evidence that these RNAs have been shaped by either direct or congruent selection for any kind of robustness. Instead, the high robustness of Drosophila pre-miRNAs appears to be mostly intrinsic and likely a consequence of selection for functional structures.

FIG. 1.

No evidence of evolution of mutational robustness in Drosophila pseudoobscura mir-317. (A) Secondary structure of dps-mir-317. The pre-miRNA folds into a stem–loop hairpin structure. The horizontal line marks the location of the mature miRNA. (B) Density plot of the structure-constrained null distribution of mutational robustness for dps-mir-317. The null distribution consists of 1000 sequences differing from the predicted ancestor in any k = 5 nucleotide positions but with exactly the same length (L = 90 nt) and structure as dps-mir-317. See supplementary figure S1 (Supplementary Material online) for additional null distributions.

FIG. 2.

Summary of the pre-miRNA data set. Seventy-one sets of orthologous pre-miRNA genes from Drosophila were analyzed using the above phylogenetic tree (Siepel et al. 2005; Rosenbloom et al. 2010). (See http://tinyurl.com/drostree for original.) Divergence dates were taken from (Tamura et al. 2004). Only evolutionary events that occurred on the terminal branches (black lines) were counted. For genes found in all 12 species, inferred ancestors were used at the nodes with black circles. The table summarizes the number of branches/sequences for each species that were filtered for (1) showing no evolution, (2) containing indels, or (3) having null distributions with low resolution, that is, fewer than 20 unique values for mutational robustness. The remaining branches were part of the final data set. See Materials and Methods for more details.

FIG. 3.

Mutational and environmental robustness have not increased from ancestor to descendant. Comparisons of mutational and environmental robustness in the 221 natural pre-miRNAs used in this study with estimated values in corresponding ancestors. Numbers in the bottom left of panels indicate the percentage of points in either half of the panel, defined by the y = x diagonal. Some outlying points (<10) are not plotted (see supplementary fig. S2, Supplementary Material online).

FIG. 4.

Neutral evolution of robustness. (A) The CDF of q values (black lines) of the robustness of pre-miRNAs compared with their corresponding structure-constrained null distributions. Anderson-Darling test statistics (A) and their associated P values are also shown. The dashed lines represent the expected values of points in a CDF for a uniform distribution, and dotted lines mark 95% concentration bands (only 5% of uniform CDFs of this size are expected to have at least one point outside this region). (B) Histograms of the simulated distributions for each A statistic; P values do not change noticeably.

FIG. 5.

Strong purifying selection against changes in secondary structure. (A) CDF of q values of the robustness of pre-miRNAs compared with their corresponding structure-unconstrained null distributions. (B) CDF of q values of the structural distance between ancestor and descendant pre-miRNAs compared with their corresponding structure-unconstrained null distributions. (C) Median mutational robustness of the sequences from the structure-unconstrained null distributions binned according to their structural distance from the natural descendant pre-miRNA. Robustness decreases as the distance to natural structures increases.