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1. An Unconventional Flavivirus and Other RNA Viruses in the Sea Cucumber (Holothuroidea; Echinodermata) Virome

   https://doi.org/10.3390/v12091057  

   Hewson, Ian; Johnson, Mitchell R.; Tibbetts, Ian R. (September 2020, Viruses)

   null (Ed.)

   Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting eight species (representing four families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a hitherto undocumented wasting disease. The RNA virome comprised genome fragments of both single-stranded positive sense and double stranded RNA viruses, including those assigned to the Picornavirales, Ghabrivirales, and Amarillovirales. We discovered an unconventional flavivirus genome fragment which was most similar to a shark virus. Ghabivirales-like genome fragments were most similar to fungal totiviruses in both genome architecture and homology and had likely infected mycobiome constituents. Picornavirales, which are commonly retrieved in host-associated viral metagenomes, were similar to invertebrate transcriptome-derived picorna-like viruses. The greatest number of viral genome fragments was recovered from the wasting A. californicus library compared to the asymptomatic A. californicus library. However, reads from the asymptomatic library recruited to nearly all recovered wasting genome fragments, suggesting that they were present but not well represented in the grossly normal specimen. These results expand the known host range of flaviviruses and suggest that fungi and their viruses may play a role in holothurian ecology. 

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2. Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions

   https://doi.org/10.1038/s41467-021-21039-0  

   Nichols, Parker J.; Bevers, Shaun; Henen, Morkos; Kieft, Jeffrey S.; Vicens, Quentin; Vögeli, Beat (December 2021, Nature Communications)

   null (Ed.)

   Abstract Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1. 

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3. Insecticidal RNA interference, thinking beyond long dsRNA

   https://doi.org/10.1002/ps.6147  

   Flynt, Alex_S (November 2020, Pest Management Science)

   Abstract Over 20 years ago double‐stranded RNA (dsRNA) was described as the trigger of RNAi interference (RNAi)‐based gene silencing. This paradigm has held since, especially for insect biopesticide technologies where dsRNAs, similar to those described in 1998, are used to inhibit gene expression. In the intervening years, investigation of RNAi pathways has revealed the small RNA effectors of RNAi are diverse and rapidly evolving. The rich biology of insect small RNAs suggests potential to use multiple RNAi modes for manipulating gene expression. By exploiting different RNAi pathways, the menu of options for pest control can be expanded and could lead to better tailored solutions. Fortunately, basic delivery strategies used for dsRNA such as direct application or transgenic expression will translate well between RNAs transiting different RNAi pathways. Importantly, further engineering of RNAi‐based biopesticides may provide an opportunity to address dsRNA insensitivity seen in some pests. Characterization of RNAi pathways unique to target species will be indispensable to this end and may require thinking beyond long dsRNA. © 2020 Society of Chemical Industry 

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4. Phylogenetic analyses of seven protein families refine the evolution of small RNA pathways in green plants

   https://doi.org/10.1093/plphys/kiad141  

   Bélanger, Sébastien; Zhan, Junpeng; Meyers, Blake C. (March 2023, Plant Physiology)

   Abstract Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways. 

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5. Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

   Zaharias, S.; Zhang, Z.; Davis, K.; Fargason, T.; Cashman, D.; Yu, T.; Zhang, J. (July 2021, Journal of biological chemistry)

   Musier-Forsyth, Karin (Ed.)

   RNA-binding proteins play crucial roles in various cellular functions, and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15) as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3 (SRSF3). Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, while underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts. 

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