Increased Affinity for RNA Targets Evolved Early in Animal and Plant Dicer Lineages through Different Structural Mechanisms

Abstract

Understanding the structural basis for evolutionary changes in protein function is central to molecular evolutionary biology and can help determine the extent to which functional convergence occurs through similar or different structural mechanisms. Here, we combine ancestral sequence reconstruction with functional characterization and structural modeling to directly examine the evolution of sequence-structure-function across the early differentiation of animal and plant Dicer/DCL proteins, which perform the first molecular step in RNA interference by identifying target RNAs and processing them into short interfering products. We found that ancestral Dicer/DCL proteins evolved similar increases in RNA target affinities as they diverged independently in animal and plant lineages. In both cases, increases in RNA target affinities were associated with sequence changes that anchored the RNA’s 5′phosphate, but the structural bases for 5′phosphate recognition were different in animal versus plant lineages. These results highlight how molecular-functional evolutionary convergence can derive from the evolution of unique protein structures implementing similar biochemical mechanisms.

Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites

“…To further investigate the recognition of dsRNAs by the Dicer PPC domain, we constructed PPC domains of human Dicer with 3′ or 5′ pocket mutations and tested their affinity for dsRNAs. We measured protein–RNA binding using a label-free in vitro kinetic assay in which the biotinylated dsRNA duplexes were bound to streptavidin probes and exposed to various concentrations of recombinant human Dicer PPC domains ( 39 ). We generated miR-HSUR4-5p with or without +15 nt extension by in vitro transcription, and annealed them with a biotinylated miR-HSUR4-3p to produce dsRNA duplexes ( Supplementary Figure S11 ).…”

Origin and evolution of the plant immune system

“…), indicating that RNAi is not essential in all eukaryotes, especially unicellular organisms (Shabalina & Koonin, ; Cerutti et al ., ). Four distinct DCL groups (DCL‐1 to DCL‐4) seem to have arisen in the early evolution of land plants (Mukherjee et al ., ; Jia et al ., ). Recurrent positive selection occurred in DCL proteins throughout plants, especially in DCL‐4 proteins that mainly mediate antiviral RNA silencing in plants (Mukherjee et al ., ; Jia et al ., ).…”

“…Four distinct DCL groups (DCL‐1 to DCL‐4) seem to have arisen in the early evolution of land plants (Mukherjee et al ., ; Jia et al ., ). Recurrent positive selection occurred in DCL proteins throughout plants, especially in DCL‐4 proteins that mainly mediate antiviral RNA silencing in plants (Mukherjee et al ., ; Jia et al ., ). Ancestral sequence resurrection suggests that the ancestral plant DCL protein exhibits affinity for viral RNA (Jia et al ., ).…”

“…Recurrent positive selection occurred in DCL proteins throughout plants, especially in DCL‐4 proteins that mainly mediate antiviral RNA silencing in plants (Mukherjee et al ., ; Jia et al ., ). Ancestral sequence resurrection suggests that the ancestral plant DCL protein exhibits affinity for viral RNA (Jia et al ., ). The affinity for viral RNA was increased during the evolution of DCL‐4 proteins and was increased and then decreased during the evolution of DCL‐2s (Jia et al ., ).…”

“…Ancestral sequence resurrection suggests that the ancestral plant DCL protein exhibits affinity for viral RNA (Jia et al ., ). The affinity for viral RNA was increased during the evolution of DCL‐4 proteins and was increased and then decreased during the evolution of DCL‐2s (Jia et al ., ). Indeed, antiviral RNA silencing is mediated by DCL‐4 and probably DCL‐2 in some conditions (Ding, ).…”