Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses

Rozen-Gagnon, Kathryn; Gu, Meigang; Luna, Joseph M.; Luo, Ji‐Dung; Yi, Soon; Novack, Sasha; Jacobson, Eliana; Wang, Wei; Paul, Matthew R.; Scheel, Troels K. H.; Carroll, Thomas; Rice, Charles M.

doi:10.1016/j.chom.2021.03.004

Cited by 10 publications

(11 citation statements)

References 91 publications

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“…Such gene knock-ins, while highly desirable for detailed analysis of the function of genomic regions, are more difficult to achieve than gene knock-outs because of the low efficiency of HDR in eukaryotes (Hagmann et al, 1998). Although success with HDR has been reported in some insects including the silk moth Bombyx mori (Ma et al, 2014; Zhu et al, 2015), multiple mosquito species (Hammond et al, 2016; Kistler et al, 2015; Purusothaman et al, 2021) and mosquito cell lines (Rozen-Gagnon et al, 2021), the beetle Tribolium castaneum (Gilles et al, 2015), the medfly Ceratitis capitata (Aumann et al, 2018), and the squinting bush brown butterfly Bicyclus anynana (Connahs et al, 2022), our attempts at HDR-based knock-in techniques have never succeeded in G. bimaculatus (unpublished observations). Recently, an efficient gene knock-in method through NHEJ was developed in the zebrafish Danio rerio (Auer et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

Matsuoka

Nakamura

Watanabe

et al. 2021

Preprint

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Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

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Section: Introductionmentioning

confidence: 99%

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

Matsuoka

Nakamura

Watanabe

et al. 2021

Preprint

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“…This pattern is reminiscent of 3' UTR biased mapping observed for the other cISFs, CxFV, CFAV and AEFV, although the skewed distribution is much more pronounced for KRV (Fig. 4A-B) (2,46,60). About 14% of the vsiRNAs of CxFV and CFAV and more than 95% of KRV vsiRNAs derived from their 3' UTRs, in stark contrast to the other flaviviruses for which a median of ~4% of vsiRNA mapped to the 3' UTR (Fig.…”

Section: Asymmetric Distribution Of Vsirnas Across the Krv Genomementioning

confidence: 69%

Pan-flavivirus analysis reveals sfRNA-independent, 3’UTR-biased siRNA production from an Insect-Specific Flavivirus

Besson¹,

Overheul

Wolfinger

et al. 2022

Preprint

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Flaviviruses subvert the host RNA decay machinery to produce subgenomic flavivirus RNA (sfRNA), products of the 5’-3’ exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3’ untranslated region (UTR) of the viral genome. The classical insect-specific flavivirus (cISF) Kamiti River virus (KRV) has a unique 1.2 kb long 3’ UTR, of which only 40% is homologous to its closest family member, cell-fusing agent virus (CFAV). We mapped the 5’ end of KRV sfRNAs and found that KRV produces high copy numbers of a long, 1017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Furthermore, we identified a new positive and negative sense 1.5 kb subgenomic RNA species that is colinear with the 3’ region of the NS5 gene and the viral 3’ UTR, which we tentatively named subgenomic cISF RNA (cifRNA). Expression of both sfRNA1 and sfRNA2 was reduced in Pacmandeficient Aedes albopictus mosquito cells, while expression of the longer cifRNA was Pacman-independent. Interestingly, a pan-Flavivirus small RNA analysis in Aedes albopictus cells revealed that nearly all KRV-derived siRNAs mapped to the 3’ UTR region and that these siRNAs are produced in high quantity. 3’ UTR-biased siRNA production appeared to be conserved in other cISFs, albeit to a lesser extent, whereas siRNAs were evenly distributed across the viral genome of other representatives of the Flavivirus genus. We suggest that cISFs and particularly KRV developed a unique mechanism to produce high amounts of siRNA as a decoy of the antiviral RNAi response.

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“…We performed coCLIP and created sequencing libraries from nuclear, cytosolic, or G3BP tethered APEX2 under mock or sodium arsenite conditions, all with associated input and enriched libraries, for 58 sequenced libraries in total ( Table S1 ). To facilitate robust and consistent data analysis, similar to previous efforts for CLIP in non-model organisms, 25 we further created a custom single command-line analysis pipeline, termed “CLIPpityCLIP,” to process raw sequencing data into peaks for subsequent analysis and visualization ( Figure S3 , see methods). We first compared nuclear and cytoplasmic coCLIP data to input HuR-CLIP from the same samples as a reference, all under unstressed conditions.…”

Section: Resultsmentioning

confidence: 99%

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP

Yi,

Singh,

Rozen-Gagnon

et al. 2023

Preprint

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RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

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Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses

Cited by 10 publications

References 91 publications

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

Pan-flavivirus analysis reveals sfRNA-independent, 3’UTR-biased siRNA production from an Insect-Specific Flavivirus

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP

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