Function and Evolution of Nematode RNAi Pathways

Abstract

Selfish genetic elements, like transposable elements or viruses, are a threat to genomic stability. A variety of processes, including small RNA-based RNA interference (RNAi)-like pathways, has evolved to counteract these elements. Amongst these, endogenous small interfering RNA and Piwi-interacting RNA (piRNA) pathways were implicated in silencing selfish genetic elements in a variety of organisms. Nematodes have several incredibly specialized, rapidly evolving endogenous RNAi-like pathways serving such purposes. Here, we review recent research regarding the RNAi-like pathways of Caenorhabditis elegans as well as those of other nematodes, to provide an evolutionary perspective. We argue that multiple nematode RNAi-like pathways share piRNA-like properties and together form a broad nematode toolkit that allows for silencing of foreign genetic elements.

Keywords: 21U RNA; 22G RNA; 26G RNA; Argonaute; C. elegans; Piwi; RdRP; nematode; piRNA; siRNA; small RNA.

Figure 1

Phylogenetic tree depicting the three main eukaryotic AGO clades. To provide a broad phylogenetic representation across eukaryotes, the AGO proteins of C. elegans were aligned with those of human, fruit fly, fission yeast and starlet sea anemone. For tractability, we chose not to include plant AGOs. Two prokaryotic AGOs were used to root the tree. A multiple sequence alignment of AGO sequences was constructed using MUSCLE [22]. The phylogenetic tree was constructed with ClustalW [23], excluding positions with gaps and represented with NJPlot [24]. Numbers indicate bootstrapping values. The sequences and the alignment are provided as Supplementary Files. AGOs are represented by a two letter prefix indicating the species, followed by the AGO name or UniProt ID. Species legend: Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mp, Marinitoga piezophila; Nv, Nematostella vectensis; Rs, Rhodobacter sphaeroides; Sp, Schizosaccharomyces pombe.

Figure 2

Biogenesis and function of the PRG-1/21U-RNA pathway and its antagonistic signals. (A) In the C. elegans germline, the USTC complex, with SNPC-4, PRDE-1 and TOFU-4/5, recognize the upstream motif and promote 21U-RNA transcription by RNA PolII. 21U-RNAs are exported to the cytoplasm for further processing. 5′ processing may be undertaken by the PETISCO complex, whereas PARN trims the 3′ ends of 21U-RNAs. 21U-RNAs subsequently associate with PRG-1 and are 2′-O-methylated at the 3′ end by HENN-1. PRG-1 scans the germline transcriptome and stimulates 22G-RNA production, by RdRPs and mutator factors, from foreign transcripts. Then, 22G-RNAs bind to cytoplasmic WAGOs, leading to PTGS of targets or to HRDE-1, which promotes TGS. Accessory NRDE factors are required for HMT recruitment and deposition of repressive chromatin marks. Moreover, NRDE factors may directly impair the activity of RNA PolII. (B) CSR-1 and PATCs inhibit repressive PRG-1 signals. The regulatory outcome of a germline-expressed gene will depend on the balance between associated CSR-1/22G-RNAs and PRG-1/21U-RNAs. Germline genes are strongly recognized by CSR-1 and have PATCs throughout their sequence, thereby inhibiting PRG-1 targeting. In contrast, non-self sequences tend to lack the PATC signature and will not be strongly targeted by CSR-1. In this case, stronger PRG-1 targeting will flag these transcripts for degradation. HMT, Histone methyltransferases; PolII, RNA Polymerase II; PTGS, post-transcriptional gene silencing; RdRP, RNA-dependent RNA Polymerase; RNAa, RNA-induced epigenetic gene activation; RNAe, RNA-induced epigenetic silencing; TGS, transcriptional gene silencing.

Figure 3

Biogenesis and function of 26G-RNA pathways. Two distinct subpopulations of 26G-RNAs are produced by the ERI complex, which includes the RdRP RRF-3 and DCR-1. To form a mature ERI complex, ERI-5 and GTSF-1 tether RRF-3 to the other ERI complex factors. One subpopulation of 26G-RNAs is produced in the male germline and in the spermatogenic gonad in L4 hermaphrodites (on the left). These 26G-RNAs associate with ALG-3/4 and target spermatogenic transcripts. This is thought to trigger the biogenesis of 22G-RNAs that interact with CSR-1, providing a transgenerational memory of paternal gene expression. In addition, such ALG-3/4-triggered 22G-RNAs may associate with other WAGOs and negatively regulate target gene expression. Another subpopulation of 26G-RNAs is expressed in the oogenic gonad and in embryos (on the right). These associate with ERGO-1 and are 2′-O-methylated by HENN-1 at their 3′ end. ERGO-1-bound 26G-RNAs target a diverse set of genes, which tend to be short, with few introns and with weak splicing signals. ERGO-1 triggers 22G-RNA biogenesis, which in turn can promote PTGS through their association with cytoplasmic WAGOs, as well as TGS through association with the nuclear WAGO protein NRDE-3. NRDE-3, with the assistance of additional NRDE factors and HMTs, perpetuates silencing of 26G-RNA targets in the soma throughout animal development. Of note, NRDE-3 acts in a manner analogous to HRDE-1 in the germline (see Figure 2A). HMTs, Histone methyltransferases; PTGS, post-transcriptional gene silencing; RdRP, RNA-dependent RNA Polymerase; TGS, transcriptional gene silencing.

Figure 4

Phylogenetic analysis of the MID, PAZ and PIWI domains of AGO proteins. Phylogenetic trees of the MID (A), PAZ (B) and PIWI (C) domains of AGO proteins. ERGO-1 is represented in bold. The multiple sequence alignment used in Figure 2 was trimmed to the coordinates of either the MID, the PAZ or the PIWI domains, respectively and each tree was constructed as in Figure 2. The alignments are provided as Supplementary Files. AGOs are represented by a two letter prefix indicating the species, followed by the AGO name or UniProt ID. Monophyletic groups including most of the members of one clade are grouped by color. Species legend: Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mp, Marinitoga piezophila; Nv, Nematostella vectensis; Rs, Rhodobacter sphaeroides; Sp, Schizosaccharomyces pombe.