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1. Small RNAs >26 nt in length associate with AGO1 and are upregulated by nutrient deprivation in the alga Chlamydomonas

   https://doi.org/10.1093/plcell/koad093  

   Li, Yingshan; Kim, Eun-Jeong; Voshall, Adam; Moriyama, Etsuko N; Cerutti, Heriberto (March 2023, The Plant Cell)

   Abstract Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks. 

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2. Message in a Bubble: Shuttling Small RNAs and Proteins Between Cells and Interacting Organisms Using Extracellular Vesicles

   https://doi.org/10.1146/annurev-arplant-081720-010616  

   Cai, Qiang; He, Baoye; Wang, Shumei; Fletcher, Stephen; Niu, Dongdong; Mitter, Neena; Birch, Paul R.; Jin, Hailing (June 2021, Annual Review of Plant Biology)

   Communication between plant cells and interacting microorganisms requires the secretion and uptake of functional molecules to and from the extracellular environment and is essential for the survival of both plants and their pathogens. Extracellular vesicles (EVs) are lipid bilayer–enclosed spheres that deliver RNA, protein, and metabolite cargos from donor to recipient cells and participate in many cellular processes. Emerging evidencehas shown that both plant and microbial EVs play important roles in cross-kingdom molecular exchange between hosts and interacting microbes to modulate host immunity and pathogen virulence. Recent studies revealed that plant EVs function as a defense system by encasing and delivering small RNAs (sRNAs) into pathogens, thereby mediating cross-species and cross-kingdom RNA interference to silence virulence-related genes. This review focuses on the latest advances in our understanding of plant and microbial EVs and their roles in transporting regulatory molecules, especially sRNAs, between hosts and pathogens. EV biogenesis and secretion are also discussed, as EV function relies on these important processes. 

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3. Characterization of a novel sRNA contributing to biofilm formation in Salmonella enterica serovar Typhimurium

   https://doi.org/10.17912/micropub.biology.000796  

   Naaz, Sayema; Sakib, Najmuj; Houserova, Dominika; Badve, Rani; Crucello, Aline; Borchert, Glen M (January 2023, microPublication Biology)

   Small RNAs (sRNAs) are short noncoding RNAs of ~50-200 nucleotides believed to primarily function in regulating crucial activities in bacteria during periods of cellular stress. This study examined the relevance of specific sRNAs on biofilm formation in nutrient starved Salmonella enterica serovar Typhimurium. Eight unique sRNAs were selected for deletion primarily based on their genomic location and/or putative targets. Quantitative and qualitative analyses confirm one of these, sRNA1186573, is required for efficient biofilm formation in S. enterica further highlighting the significance of sRNAs during Salmonella stress response. 

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4. RNA ‐based fluorescent biosensors for live cell detection of bacterial sRNA

   https://doi.org/10.1002/bip.23394  

   Kitto, Rebekah Z.; Christiansen, Kylee E.; Hammond, Ming C. (August 2020, Biopolymers)

   Abstract Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up‐ or downregulation of protein synthesis.In vivoimaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limitedin vivoand do not provide real‐time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA‐based fluorescent biosensor for bacterial sRNA bothin vitroandin vivo. We validated these sensors for three different bacterial sRNAs inEscherichia coliand demonstrated that the designs provide a bright, sequence‐specific signal output in response to exogenous and endogenous RNA targets. 

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5. Characterization of 475 Novel, Putative Small RNAs (sRNAs) in Carbon-Starved Salmonella enterica Serovar Typhimurium

   https://doi.org/10.3390/antibiotics10030305  

   Houserova, Dominika; Dahmer, Donovan J.; Amin, Shivam V.; King, Valeria M.; Barnhill, Emmaline C.; Zambrano, Mike E.; Dean, Meghan A.; Crucello, Aline; Aria, Kevin M.; Spector, Michael P.; et al (March 2021, Antibiotics)

   Neuwirth, Catherine (Ed.)

   An increasingly apparent role of noncoding RNA (ncRNAs) is to coordinate gene expression during environmental stress. A mounting body of evidence implicates small RNAs (sRNAs) as key drivers of Salmonella stress survival. Generally thought to be 50–500 nucleotides in length and to occur in intergenic regions, sRNAs typically regulate protein expression through base pairing with mRNA targets. In this work, through employing a refined definition of sRNAs allowing for shorter sequences and sRNA loci to overlap with annotated protein-coding gene loci, we have identified 475 previously unannotated sRNAs that are significantly differentially expressed during carbon starvation (C-starvation). Northern blotting and quantitative RT-PCRs confirm the expressions and identities of several of these novel sRNAs, and our computational analyses find the majority to be highly conserved and structurally related to known sRNAs. Importantly, we show that deletion of one of the sRNAs dynamically expressed during C-starvation, sRNA4130247, significantly impairs the Salmonella C-starvation response (CSR), confirming its involvement in the Salmonella CSR. In conclusion, the work presented here provides the first-ever characterization of intragenic sRNAs in Salmonella, experimentally confirms that sRNAs dynamically expressed during the CSR are directly involved in stress survival, and more than doubles the Salmonella enterica sRNAs described to date. 

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