Evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral RNA binding in plants

Abstract

RNA interference (RNAi) is a eukaryotic molecular system that serves two primary functions: 1) gene regulation and 2) protection against selfish elements such as viruses and transposable DNA. Although the biochemistry of RNAi has been detailed in model organisms, very little is known about the broad-scale patterns and forces that have shaped RNAi evolution. Here, we provide a comprehensive evolutionary analysis of the Dicer protein family, which carries out the initial RNA recognition and processing steps in the RNAi pathway. We show that Dicer genes duplicated and diversified independently in early animal and plant evolution, coincident with the origins of multicellularity. We identify a strong signature of long-term protein-coding adaptation that has continually reshaped the RNA-binding pocket of the plant Dicer responsible for antiviral immunity, suggesting an evolutionary arms race with viral factors. We also identify key changes in Dicer domain architecture and sequence leading to specialization in either gene-regulatory or protective functions in animal and plant paralogs. As a whole, these results reveal a dynamic picture in which the evolution of Dicer function has driven elaboration of parallel RNAi functional pathways in animals and plants.

Fig. 1.

Dicer protein consists of multiple RNA-interacting functional domains. We plot the domain architecture of human Dicer along the protein sequence; other Dicer proteins from animals and plants have similar domain architectures (figs. 2 and 4). Functional domains were identified via sequence searches of the PFam database (Punta et al. 2011), the SMART database (Letunic et al. 2012) and the NCBI conserved-domain database (Marchler-Bauer et al. 2011). The 5′ RNA-binding pocket extension of the canonical PAZ domain found in some animal Dicers is also shown (Park et al. 2011).

Fig. 2.

Phylogenetic analysis supports independent expansions of an ancient eukaryote Dicer protein in animals and plants. We plot the support for monophyletic expansions of Dicer paralogs in animals and plants. Support is given as SH-like aLRT scores/maximum-likelihood bootstrap proportions/Bayesian posterior probabilities. See supplementary figures S1–S5, Supplementary Material online, for full trees and additional analyses.

Fig. 3.

Dicer duplicated early in animal evolution. We inferred the metazoan Dicer family phylogeny using maximum likelihood and Bayesian methods. Support is shown for key nodes as SH-like aLRT scores/bootstrap proportions/Bayesian posterior probabilities (supplementary figs. S6–S11, Supplementary Material online, for additional support calculations and analyses). We also show the inferred domain architecture for each sequence. We used a branch-sites model to identify protein-coding adaptation from aligned codon sequences (Zhang et al. 2005). Colored circles indicate branches showing significant support for adaptation in each functional domain (P < 0.05 after correcting for multiple tests). Species names are colored by taxonomic group.

Fig. 4.

Dicer duplicated early in plant evolution. We inferred the plant Dicer family phylogeny using maximum likelihood and Bayesian methods. Support is shown as SH-like aLRT scores / bootstrap proportions / Bayesian posterior probabilities (supplementary figs. S13–S17, Supplementary Material online). We additionally display the inferred domain architecture for each sequence. Colored circles on each branch indicate significant support (P < 0.05 after correcting for multiple tests) for adaptive protein-coding changes in each functional domain, inferred using branch-sites analysis (Zhang et al. 2005). Species names are colored by taxonomic group.

Fig. 5.

Adaptive substitutions altered the electrostatic distribution across the RNA-binding pocket of plant DCL-4 PAZ. We inferred the 3D structure and electrostatic distribution of plant and animal Dicer PAZ domains (see Materials and Methods). We plot the electrostatic distribution (kT/e) across the RNA-binding pocket (yellow-dotted outline) of each protein.

Fig. 6.

Adaptive substitutions in plant DCL-4 PAZ domain are likely to affect RNA binding. We inferred adaptive protein-coding substitutions in monocot and dicot DCL-4 PAZ domains using branch-sites models (see Materials and Methods). We plot adaptive substitutions (posterior probability > 0.95) along the protein structure (top) and multiple sequence alignment (bottom). RNA-contacting residues identified in Giardia Dicer PAZ (Simon et al. 2011) and AGO PAZ (Wang, Juranek, et al. 2009) are indicated below the sequence alignment.

Fig. 7.

Examination of Dicer sequence alignment reveals bilaterian- and DCL-1-specific N-terminal PAZ extensions likely to contribute to 5′ RNA-binding. We display parts of aligned Dicer sequences from representative taxa. Protein domain architecture is show in the middle, with the PAZ domain indicated in yellow. Specific residues previously shown to contribute to 3′ and 5′ RNA-binding are indicated at the top of each alignment (Lingel et al. 2004; Wang, Juranek, et al. 2009; Park et al. 2011; Simon et al. 2011). Alignment of conserved N-terminal PAZ extensions from bilateria and plant DCL-1 are indicated in pink and orange, respectively.