Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in<i>Aedes aegypti</i>mosquitoes

Joosten, Joep; Overheul, Gijs J.; Rij, Ronald P. van; Miesen, Pascal

doi:10.1093/nar/gkab640

Cited by 19 publications

(11 citation statements)

References 60 publications

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“…This results in piRNA sharp ends and a so-called +1U bias. Importantly, analysis of vpiRNAs from Aag2 cells infected with the alphavirus Sindbis (SINV) had suggested a Zucchini-like biogenesis mechanism in vpiRNA 3’ termini formation, as sharp ends and +1U bias were observed [ 52 ]. This led us to further investigate the origins of 21 nt vsi-piRNAs by subjecting vpiRNAs (25–30 nt) and 21 nt vsi-piRNAs sequences to a similar analysis.…”

Section: Resultsmentioning

confidence: 99%

“…They may be products of vpiRNA degradation, perhaps post-processing by the recently identified Ae . aegypti ortholog of Zucchini, and/or by the corresponding ortholog Nibbler which has 3’-5’ exonuclease activity [ 52 ]. However, this would require further studies, such as silencing the expression of these proteins followed by small RNA sequence analysis.…”

Section: Discussionmentioning

confidence: 99%

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Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

et al. 2022

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The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

et al. 2022

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“…aegypti , Piwi5 and Ago3 are the core components of the ping-pong amplification mechanism that generates vpiRNAs following Sindbis virus infection [ 107 ]. Piwi4, on the other hand, was not found to be required for the ping-pong amplification mechanism [ 110 ]. Interestingly, knockdown of Piwi4 can result in an increase of production of primary/initiator piRNAs from piRNA clusters that is controlled by Piwi5, indicating competition between the actions of both Piwi proteins [ 110 ].…”

Section: Insights From Research On Piwi Proteins and Pirnas In Mosqui...mentioning

confidence: 99%

What Are the Functional Roles of Piwi Proteins and piRNAs in Insects?

Santos

Feng

Κολλιοπούλου

et al. 2023

Insects

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Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered.

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“…Whereas secondary piRNAs are produced only in germ cells by secondary expansion of Argonaute 3 (AGO3) protein with Aub-piRISCs. This process is also known as the ping-pong pathway [ 6 , 9 , 10 , 11 ]. Recent studies demonstrated that piRNAs, as newly emerging regulators of gene expression, could exert functions in an array of biological processes such as immune response and development [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Transcriptome-Wide Characterization of piRNAs during the Developmental Process of European Honey-Bee Larval Guts

Long

Fan

et al. 2022

Genes

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piRNAs play pivotal roles in maintaining genome stability, regulating gene expression, and modulating development and immunity. However, there are few piRNA-associated studies on honey-bees, and the regulatory role of piRNAs in the development of bee guts is largely unknown. Here, the differential expression pattern of piRNAs during the developmental process of the European honey-bee (Apis mellifera) larval guts was analyzed, followed by investigation of the regulatory network and the potential function of differentially expressed piRNAs (DEpiRNAs) in regulating gut development. A total of 843 piRNAs were identified in the larval guts of A. mellifera; among these, 764 piRNAs were shared by 4- (Am4 group), 5- (Am5 group), and 6-day-old (Am6 group) larval guts, while 11, 67, and one, respectively, were unique. The first base of piRNAs in each group had a cytosine (C) bias. Additionally, 61 up-regulated and 17 down-regulated piRNAs were identified in the “Am4 vs. Am5” comparison group, further targeting 9, 983 genes, which were involved in 50 GO terms and 142 pathways, while two up-regulated and five down-regulated piRNAs were detected in the “Am5 vs. Am6” comparison group, further targeting 1, 936 genes, which were engaged in 41 functional terms and 101 pathways. piR-ame-742536 and piR-ame-856650 in the “Am4 vs. Am5” comparison group as well as piR-ame-592661 and piR-ame-31653 in the “Am5 vs. Am6” comparison group were found to link to the highest number of targets. Further analysis indicated that targets of DEpiRNAs in these two comparison groups putatively regulate seven development-associated signaling pathways, seven immune-associated pathways, and three energy metabolism pathways. Moreover, the expression trends of five randomly selected DEpiRNAs were verified based on stem-loop RT-PCR and RT-qPCR. These results were suggestive of the overall alteration of piRNAs during the larval developmental process and demonstrated that DEpiRNAs potentially modulate development-, immune-, and energy metabolism-associated pathways by regulating the expression of corresponding genes via target binding, further affecting the development of A. mellifera larval guts. Our data offer a novel insight into the development of bee larval guts and lay a basis for clarifying the underlying mechanisms.

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

Cited by 19 publications

References 60 publications

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

What Are the Functional Roles of Piwi Proteins and piRNAs in Insects?

Transcriptome-Wide Characterization of piRNAs during the Developmental Process of European Honey-Bee Larval Guts

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