The Evolutionary Loss of RNAi Key Determinants in Kinetoplastids as a Multiple Sporadic Phenomenon

Abstract

We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.

Keywords: Evolutionary loss of genes; Kinetoplastea; RNAi; Trypanosomatida.

Fig. 1

Structural conservation of Argonaute proteins within kinetoplastid strains. AGO1 protein sequences from nine trypanosomatid strains aligned with the corresponding sequence from T. thermophilus and superimposed on its 3D model derived from the crystal structure. Conservation levels shown in different colors. The proposed nucleic-acid-binding pocket is indicated

Fig. 2

Structural conservation of Dicer1 proteins within kinetoplastid strains. Dicer1 protein sequences from nine trypanosomatid strains aligned with the corresponding sequence (human) and superimposed on its 3D model derived from the crystal structure. Conservation levels shown in different colors. Arrows point at the proposed nuclease core (yellow) and PAZ-like or a platform-like domain (blue)

Fig. 3

Evolutionary trees of trypanosomatid RNAi-related genes Argonaute (AGO1) and Dicer 1 (DCL1) genes. Molecular trees were generated as described in Materials and Methods and include gene sequences from seven sequenced trypanosomatids plus two gene sequences each from L. braziliensis and C. fasciculata, used as probes. Numbers on nodes are bootstrap support values (50 or greater shown)

Fig. 4

Evolutionary trees based on trypanosomatid housekeeping genes APRT(adenine phosphoribosyl transferase), GSH1 (γ-glutamylcysteine synthetase), and PTR1 (pteridine reductase 1). The scale corresponds to the degree of evolutionary divergence among these organisms. Lineages lacking RNAi genes are colored red, while RNAi-proficient lineages are colored blue