Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence

Abstract

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute and Dicer as essential cofactors that process and present small RNAs to their targets. Well-validated small RNA pathway cofactors such as these show distinctive patterns of conservation or divergence in particular animal, plant, fungal and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or short interfering RNA (siRNA)-mediated repression in Caenorhabditis elegans and Drosophila melanogaster, and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction data sets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about one-half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small-RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in those species.

Figure 1. Phylogenetic profiling analysis shows correlated conservation patterns ofC. elegans proteins

A. Phylogenetic profiles of 10,054 conserved C. elegans proteins across 85 other eukaryotic genomes. For each C. elegans query protein, the normalized ratio of the blastp score for the top scoring protein sequence similarity is listed in the column corresponding to each genome. Values range from 0 (white, no similarity) to 1 (blue, 100% similarity). B. Phylogenetic profiles of validated RNAi factor RDE-1 and the 49 most correlated proteins in rank order.

Figure 2. Phylogenetic clusters of candidate small RNA pathway proteins

Validated miRNA and siRNA pathway proteins map non-randomly on the phylogenetic profile; proteins that map to the same clusters are likely to function in small RNA pathways. Left panel: Clusters enriched for validated miRNA and siRNA pathway proteins, black boxes. Darker blue represents higher protein sequence similarity. Right panel: Pairwise local protein sequence alignment of all pairs of proteins in the cluster; black represents significant similarity and white no similarity.

Figure 3. Select phylogenetic clusters enriched with hits from proteomic and functional genomic small RNA screens

A. The phylogenetic profile matrix was clustered and a Max Ratio score (MRS) was calculated for every protein in each screen; 117 proteins scored significantly in miRNA (56 genes) or siRNA (75 genes) functional genomic screens, or both (14 genes). Middle panel, black tick, hit in screens; gray tick, significant MRS. B. Blue boxes, the 23 known small RNA pathway genes identified. C. From the 117 genes predicted by the phylogenetic profile, 28 genes (blue bars) show defects in siRNA silencing (p-value < 3x10^-15).

Figure 4. Inactivation of genes implicated in RNAi pathways reanimates transgenes that are silenced by RNAi

A. Expression of scm::gfp in the seam cells of an eri-1(mg366) mutant, where it is normally silenced by RNAi. Animals shown were treated with control, dcr-1, arp-6, or B0336.3 RNAi. B. GFP expression from the ubl-1::gfp-siR-1 sensor transgene, which is normally silenced by the siR-1 endogenous siRNA. Animals shown were treated with control, dcr-1, arp-6, or mes-4 RNAi.