New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing

Abstract

Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently known small RNA classes and place them in context of the reconstructed evolutionary history of the RNA interference (RNAi) protein machinery. This synthesis indicates that the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: (1) sense-antisense transcriptional products, (2) genome-encoded, imperfectly complementary hairpin sequences, and (3) larger noncoding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNAi. They were recruited alongside RNaseIII domains and RNA-dependent RNA polymerase (RdRP) domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleocytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs.

Figure 1

Temporal diagram depicting emergence of RNA substrates (left column) and core and ancillary protein domains (right column) comprising RNAi systems against the divergence of major eukaryotic lineages labeled to the left. RNAi substrates are divided according to potential for acting in the nuclear and cytoplasmic compartments. Protein domains are depicted by labeled polygonal shapes and are not drawn to scale. Names of the proteins with the depicted architectures are provided below the domains. Inferred origins for several proteins are depicted where appropriate. Affixed asterisks indicate uncertainty in terms of timing of the origins of a RNA substrate or associating protein. Affixed ampersands are indicative of a protein with a deep phylogenetic origin but potential associations with RNAi pathways in early lineages remains unexplored. Abbreviations: PNTD1, PIWI N-terminal domain 1; PNDT2, PIWI N-terminal domain 2, L2, Linker-2 domain; SNase, Staphylcoccal nuclease; nuc, nuclease; Znk, zinc knuckle; FHA, Forkhead-associated; PPlase, peptidyl prolyl-cis/trans-isomerase; Znf, zinc finger; dsRBD, double-stranded RNA-binding domain; HTH, helix-turn-helix; RdRP, RNA-dependent RNA polymerase.

Figure 2

General overview of the biogenesis of various classes of small RNA. Extremely generalized biogenesis pathways for the classes of small RNA listed in Table 2 are provided for reference. Classes of small RNA cargo are depicted and labelled numerically on the far left. Domains catalyzing or providing assistance in various lineages at steps in the pathway are shown in relevant locations. For the temporal timing of the emergence of such domains in eukaryotic evolution, please refer to Figure 1. Domains contributing to post-transcriptional modification, degradation, or amplification of small RNAs are shown to the left of each pathway. For ease in comparison, coloring of RNA and protein domains matches Figure 1. Proteins/domains which participate in RNAi pathways but cannot yet be clearly assigned to any specific pathway are not included. Core domains are labelled in white lettering.

Figure 3

Loss and expansion events of core RNAi proteins during eukaryotic evolution. Core RNAi protein components undergoing loss or extensive expansion are listed and labeled with specific affected lineages in the center columns, plotted against major eukaryotic evolutionary transitions to the left. Boxes to the far right provide more detailed resolution of loss and gain events in the kinetoplastid (bottom) and fungal/yeast lineages (top). Abbreviations: Mael, Maelstrom.

Figure 4

Structural anatomy of PIWI N-terminal domain 1 (PNTD1) and PIWI N-terminal domain 2 (PNTD2). (A) Cartoon rendering and topological diagrams of the PNTD1 and PNTD2 domains. PNTD2 has previously been referred to as the Linker-1 domain, however, as these renderings make clear, the PNTD2 domain emerged via duplication and circular permutation from the PNTD1 domain, forming a single N-terminal module which is evolutionary present across all three superkingdoms of Life and is found in architectures outside of the well-studied classical PIWI architecture depicted in the center of the figure (see also Fig. 5A). β-strands are colored in orange, α-helices are colored in purple, and extended loop regions are colored in grey. N- and C-termini are labeled with “N” and “C”, respectively. (B) Cartoon renderings of the PNTD1/PNTD2 dyad along with the N-terminal leader region containing the well-conserved asparagine residue. Three protein representatives from the three superkingdoms of Life are depicted, labeled by species and pdbid at the bottom. PNTD1 is colored in yellow, PNTD2 is colored in blue, and the leader region is colored in red. The asparagine residue is rendered as a ball and stick, colored in red, and labeled as “Asn”.

Figure 5

Genome associations and evolutionary history of the PIWI domain. (A) Domain architectures and conserved gene neighborhoods involving the PIWI domain. The inset, boxed in purple, depicts all known domain architectural themes for the PIWI domain with the phylogenies and/or PIWI families which contain these architectures provided below. Individual domains are depicted as labeled, boxed polygons. Outside of the inset, architectures and conserved gene neighborhoods containing PIWI domains and various nucleases in prokaryotes are provided. Depicted gene neighborhoods are single representatives of a cluster of sequences with the same, conserved neighborhood (for complete lists, see Further Resources). Conserved neighborhoods and domain architectures are labeled by species name and gene identifier (gi) number, separated by a semicolon. (B) Major events in the evolutionary history of the PIWI domain. Emergence of the PIWI domain from an Endonuclease V/UvrC ancestor, the likely cluster of pPIWI domains from which the eukaryotic PIWI versions emerged, and the higher-order relationships of individual PIWI families are depicted. Related groups of pPIWI class I and class II proteins are labeled in green according to the genome associations observed in (A). The group containing the recently-published PIWI-IP seq dataset from R. sphaeroides (α-helical+REase) is denoted with a blue asterisk. Predicted functional shifts for PIWI-centered systems are labeled in red. Dashed lines indicate uncertainty in terms of the origins of a lineage or timing of an event. Abbreviations: REase, Restriction endonuclease; put. nuc., putative nuclease; H-kinase, histidine kinase.