Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides

Abstract

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here, we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri , at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis . We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.

Small RNAs – Big Players in Plant-Microbe Interactions

“…The Arabidopsis ago1-27 mutant displayed reduced susceptibility to B. cinerea infection, further supporting that fungal sRNAs depend on host AGO1 to carry out their cross-kingdom RNAi function (Weiberg et al, 2013). In animal-parasite interactions, the gastrointestinal parasite nematode Heligmosomoides bakeri was recently found to secrete a specific AGO protein, exWAGO, in the EVs, which may contribute to selective export of specific siRNAs (Chow et al, 2019). Co-delivery of sRNA and AGO proteins may also facilitate sRNA stability and function, which likely increases the silencing efficiency (Li et al, 2018).…”

Different Genetic Sources Contribute to the Small RNA Population in the Arbuscular Mycorrhizal Fungus Gigaspora margarita

“…In 2013, Weiberg et al (2013) discovered that RNAi is also a key molecular component of interspecies communication: sRNAs can be transferred across the contact interface of two interacting organisms and, acting as pathogen effectors, they silence specific genes in host cells in order to favor colonization. This phenomenon, known as cross-kingdom RNAi, occurs in several pathogenic and parasitic interactions (Weiberg et al, 2013;Mayoral et al, 2014;Zhang et al, 2016;Shahid et al, 2018;Chow et al, 2019;Cui et al, 2019) where it can function as an attack or a defense strategy. Interestingly, it was also recently described in the legume-rhizobium symbiosis where bacterial transfer RNA (tRNA)-derived small RNA fragments are signal molecules that modulate host gene expression and nodule formation (Ren et al, 2019).…”

Evidence for transposable element control by Argonautes in a parasitic flatworm lacking the piRNA pathway

“…Organisms from plants 23 to humans have developed elegant molecular mechanisms to regulate TE activity. These 24 are broadly divided into epigenetics, base or histone modifications that occur at the 25 locus where the TE is encoded, and silencing or more specifically nuclear silencing (NS), 26 where transcripts arising from active TE genes are intercepted and degraded by 'host' 27 mechanisms ultimately preventing their insertion in the 'host' genome.…”

“…151 It is possible that FLAGOs have similar roles to those of the newly identified AGO in 152 nematodes. In a recent report, Chow et al [27]demonstrated that a specific nematode 153 AGO, exWAGO (extracellular WAGO), is released inside vesicles and to the environment 154 by Heligmosomoides polygyrus bakeri, a gastrointestinal nematode. The authors further 155 report that exWAGO is associated with siRNAs with sequence identity to newly evolved 156 TEs.…”

“…The localisation of SmAGO2 to the nucleus and the cytoplasm (Figure 3) may reflect 158 a dual role for this protein. While in the nucleus, SmAGO2 could undertake nuclear 159 silencing using TE-derived small RNAs [24] to target nascent TE transcripts, when 160 shifted to the cytoplasm, SmAGO2 could be packed into vesicles ready to be deployed to 161 4/10 the environment, as is the case in H. polygyrus bakeri [27]. However, proteomics studies 162 targeting extracellular vesicles have not detected SmAGO2 in samples from S. mansoni 163 [28,29,30].…”