Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>PIWI interacting (pi)RNAs are small RNAs mostly known to protect the genomes of animal germlines against transposable elements. In <jats:italic>Drosophila</jats:italic>, piRNA-mediated cleavage of transposon RNA triggers the release of a responder piRNAs via the ping-pong amplification cycle. Responder piRNA 3’ end formation by the endonuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of piRNAs that target transposons. Intriguingly, in <jats:italic>Aedes aegypti</jats:italic> mosquitoes, somatic piRNAs are produced from viral RNA, implying a role of viral (v)piRNAs in antiviral immunity. Knowledge on how vpiRNA 3’ ends are formed and whether viral RNA is subjected to trailer piRNA production, however, is lacking. To address these questions, we analyzed small RNA sequencing libraries from <jats:italic>Ae. aegypti</jats:italic> cells. We found that virus- and transposon-derived piRNAs have sharply defined 3’ ends, and that uridine residues are enriched directly downstream of dominant piRNA sequences, both of which are characteristic features of Zucchini-like cleavage of precursor piRNAs. Next, we designed a piRNA reporter system based on Sindbis virus recombinants that harbor target sites for abundant endogenous piRNAs. These piRNAs guide cleavage of the viral RNA, which is subsequently processed into abundant responder piRNAs. Using this reporter virus system, we identified the <jats:italic>Ae. aegypti</jats:italic> orthologs of Zucchini, which is required for sharp 3’ end formation of responder piRNAs, and Nibbler, a 3’-5’ exonuclease involved in the trimming of a subset of piRNAs and miRNAs. Furthermore, we found that cleavage of viral RNA triggers the production of trailer piRNAs, thus expanding the piRNA sequence pool that is able to target viral RNA. Our results have important implications for understanding how autonomous piRNA production from viral RNA can be triggered by just a few cleavage events by genome-encoded piRNAs.</jats:p>

A piRNA-lncRNA regulatory network initiates responder and trailer piRNA formation during mosquito embryonic development

“…While mismatches between guide and target RNA, especially around the slice site, are considered to abolish target RNA slicing by PIWI proteins, studies in various model systems show that low-level slicing does occur even in the absence of full complementarity around the slice site (Joosten et al 2020, Mohn et al 2015, Reuter et al 2011. In line with these findings, our data provide evidence that propiR1 directs slicing of lnc027353, despite imperfect base pairing at and around the slice site.…”

“…Surprisingly, the production of responder and trailer piRNAs from the lnc027353 cleavage fragment and the detection of slice products, illustrates that the transcript is at least in part silenced through slicing, despite the fact that the propiR1 target site in lnc027353 contains mismatches at the putative slice site. The exact sequence requirements necessary to accommodate slicing and subsequent responder piRNA production remain hitherto unexplored, but it has been observed before that slicing may occur, albeit inefficiently, in the absence of complete base pairing between small RNA and target RNA in the seed region and the slice site (Joosten et al 2020, Mohn et al 2015, Reuter et al 2011.…”

Structure-function analysis of microRNA 3′-end trimming by Nibbler

“…S1). The function of AaNbr in shaping 3ʹ-ends of small RNAs has been confirmed recently (37), establishing it as a functional ortholog of DmNbr. Recombinant AaNbr NTD (residues 26 to 405) formed a monodisperse peak on a size-exclusion column but failed to yield crystals.…”

Profile of Small RNAs, vDNA Forms and Viral Integrations in Late Chikungunya Virus Infection of Aedes albopictus Mosquitoes

“…None of these nrEVEs derive from Alphaviruses and the maximum sequence similarity between annotated nrEVE and CHIKV is 70% ( Figure 5 A). Nevertheless, nrEVEs produce piRNAs [ 35 ] and responder and trailer piRNAs are produced from viral RNAs guided by endogenous piRNAs, indicating that piRNA biogenesis can spread outside a putative nrEVE targeted region [ 31 , 101 ]. On this basis, we first verified which nrEVEs our mosquitoes had, then tested whether the piRNA profile of these nrEVEs was different in infected vs. un-infected-fed samples and finally checked for putative targets of the differentially accumulated nrEVEs-derived piRNAs.…”

“…Additionally, miRNA-target interaction may also lead to positive regulation of the target by increasing mRNA stability or action on promoter sequences [ 29 ]. Despite these differences, increasing experimental evidence reveals crosstalk among RNAi pathways, firstly between the siRNA and piRNA pathways, after arboviral infections of mosquitoes [ 30 , 31 ]. One of the links between the two pathways are viral DNA (vDNAs) fragments, which appear hours after arboviral infections and serve to amplify siRNA-mediated silencing [ 30 , 32 , 33 ].…”