The Post-Transcriptional Regulatory Protein CsrA Amplifies Its Targetome through Direct Interactions with Stress-Response Regulatory Hubs: The EvgA and AcnA Cases.

Global rewiring of bacterial gene expressions in response to environmental cues is mediated by regulatory proteins such as the CsrA global regulator from E. coli. Several direct mRNA and sRNA targets of this protein have been identified; however, high-throughput studies suggest an expanded RNA targetome for this protein. In this work, we demonstrate that CsrA can extend its network by directly binding and regulating the evgA and acnA transcripts, encoding for regulatory proteins. CsrA represses EvgA and AcnA expression and disrupting the CsrA binding sites of evgA and acnA, results in broader gene expression changes to stress response networks. Specifically, altering CsrA-evgA binding impacts the genes related to acidic stress adaptation, and disrupting the CsrA-acnA interaction affects the genes involved in metal-induced oxidative stress responses. We show that these interactions are biologically relevant, as evidenced by the improved tolerance of evgA and acnA genomic mutants depleted of CsrA binding sites when challenged with acid and metal ions, respectively. We conclude that EvgA and AcnA are intermediate regulatory hubs through which CsrA can expand its regulatory role. The indirect CsrA regulation of gene networks coordinated by EvgA and AcnA likely contributes to optimizing cellular resources to promote exponential growth in the absence of stress.

RNA-binding proteins that preferentially interact with 8-oxoG-modified RNAs: our current understanding.

Cells encounter a variety of stresses throughout their lifetimes. Oxidative stress can occur via a myriad of factors, including exposure to chemical toxins or UV light. Importantly, these stressors induce chemical changes (e.g. chemical modifications) to biomolecules, such as RNA. Commonly, guanine is oxidized to form 8-oxo-7,8-hydroxyguanine (8-oxoG) and this modification can disrupt a plethora of cellular processes including messenger RNA translation and stability. Polynucleotide phosphorylase (PNPase), heterogeneous nuclear ribonucleoprotein D (HNRPD/Auf1), poly(C)-binding protein (PCBP1/HNRNP E1), and Y-box binding protein 1 (YB-1) have been identified as four RNA-binding proteins that preferentially bind 8-oxoG-modified RNA over unmodified RNA. All four proteins are native to humans and PNPase is additionally found in bacteria. Additionally, under oxidative stress, cell survival declines in mutants that lack PNPase, Auf1, or PCBP1, suggesting they are critical to the oxidative stress response. This mini-review captures the current understanding of the PNPase, HNRPD/Auf1, PCBP1, and YB-1 proteins and the mechanism that has been outlined so far by which they recognize and interact with 8-oxoG-modified RNAs.

Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells.

Particulate matter (PM) is a ubiquitous component of air pollution that is epidemiologically linked to human pulmonary diseases. PM chemical composition varies widely, and the development of high-throughput experimental techniques enables direct profiling of cellular effects using compositionally unique PM mixtures. Here, we show that in a human bronchial epithelial cell model, exposure to three chemically distinct PM mixtures drive unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability, DNA damage responses, and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Finally, we observed that PM mixtures with higher cadmium content induced increased DNA damage and drove redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of individual cellular morphologies provides a robust, high-throughput approach to gauge the effects of environmental stressors on biological systems and score cellular susceptibilities to pollution.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Characterization of epitranscriptome reader proteins experimentally and in silico: Current knowledge and future perspectives beyond the YTH domain.

To date, over 150 chemical modifications to the four canonical RNA bases have been discovered, known collectively as the epitranscriptome. Many of these modifications have been implicated in a variety of cellular processes and disease states. Additional work has been done to identify proteins known as "readers" that selectively interact with RNAs that contain specific chemical modifications. Protein interactomes with N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (m5C), and 8-oxo-7,8-dihydroguanosine (8-oxoG) have been determined, mainly through experimental advances in proteomics techniques. However, relatively few proteins have been confirmed to bind directly to RNA containing these modifications. Furthermore, for many of these protein readers, the exact binding mechanisms as well as the exclusivity for recognition of modified RNA species remain elusive, leading to questions regarding their roles within different cellular processes. In the case of the YT-521B homology (YTH) family of proteins, both experimental and in silico techniques have been leveraged to provide valuable biophysical insights into the mechanisms of m6A recognition at atomic resolution. To date, the YTH family is one of the best characterized classes of readers. Here, we review current knowledge about epitranscriptome recognition of the YTH domain proteins from previously published experimental and computational studies. We additionally outline knowledge gaps for proteins beyond the well-studied human YTH domains and the current in silico techniques and resources that can enable investigation of protein interactions with modified RNA outside of the YTH-m6A context.

RNAs as Sensors of Oxidative Stress in Bacteria.

Oxidative stress is an important and pervasive physical stress encountered by all kingdoms of life, including bacteria. In this review, we briefly describe the nature of oxidative stress, highlight well-characterized protein-based sensors (transcription factors) of reactive oxygen species that serve as standards for molecular sensors in oxidative stress, and describe molecular studies that have explored the potential of direct RNA sensitivity to oxidative stress. Finally, we describe the gaps in knowledge of RNA sensors-particularly regarding the chemical modification of RNA nucleobases. RNA sensors are poised to emerge as an essential layer of understanding and regulating dynamic biological pathways in oxidative stress responses in bacteria and, thus, also represent an important frontier of synthetic biology.

Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells.

Particulate matter (PM) is a ubiquitous component of indoor and outdoor air pollution that is epidemiologically linked to many human pulmonary diseases. PM has many emission sources, making it challenging to understand the biological effects of exposure due to the high variance in chemical composition. However, the effects of compositionally unique particulate matter mixtures on cells have not been analyzed using both biophysical and biomolecular approaches. Here, we show that in a human bronchial epithelial cell model (BEAS-2B), exposure to three chemically distinct PM mixtures drives unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability and DNA damage responses and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization and structure, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Lastly, we observed that particulate matter mixtures with high contents of heavy metals, such as cadmium and lead, induced larger drops in viability, increased DNA damage, and drove a redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of cellular morphology provides a robust approach to gauge the effects of environmental stressors on biological systems and determine cellular susceptibilities to pollution.

CsrA Shows Selective Regulation of sRNA-mRNA Networks.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcase CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Exposure of Shewanella oneidensis MR-1 to Sublethal Doses of Ionizing Radiation Triggers Short-Term SOS Activation and Longer-Term Prophage Activation.

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (<10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria.

PNPase and RhlB Interact and Reduce the Cellular Availability of Oxidized RNA in Deinococcus radiodurans.

8-Oxo-7,8-dihydroguanine (8-oxoG) is a major RNA modification caused by oxidative stresses and has been implicated in carcinogenesis, neurodegeneration, and aging. Several RNA-binding proteins have been shown to have a binding preference for 8-oxoG-modified RNA in eukaryotes and protect cells from oxidative stress. To date, polynucleotide phosphorylase (PNPase) is one of the most well-characterized proteins in bacteria that recognize 8-oxoG-modified RNA, but how PNPase cooperates with other proteins to process oxidized RNA is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that preferably bind 8-oxoG-modified RNA in Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that preferably bind to oxidized RNA: PNPase (DR_2063), DEAD box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). Among these proteins, PNPase and RhlB exhibit high-affinity binding to 8-oxoG-modified RNA in a dose-independent manner. Deletions of PNPase and RhlB caused increased sensitivity of D. radiodurans to oxidative stress. We further showed that PNPase and RhlB specifically reduce the cellular availability of 8-oxoG-modified RNA but have no effect on oxidized DNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of pnp and rhlB compared to the single deletions. Overall, our findings suggest the roles of PNPase and RhlB in targeting 8-oxoG-modified RNAs and thereby constitute an important component of D. radiodurans resistance to oxidative stress. IMPORTANCE Oxidative RNA damage can be caused by oxidative stress, such as hydrogen peroxide, ionizing radiation, and antibiotic treatment. 8-oxo-7,8-dihydroguanine (8-oxoG), a major type of oxidized RNA, is highly mutagenic and participates in a variety of disease occurrences and development. Although several proteins have been identified to recognize 8-oxoG-modified RNA, the knowledge of how RNA oxidative damage is controlled largely remains unclear, especially in nonmodel organisms. In this study, we identified four RNA binding proteins that show higher binding affinity to 8-oxoG-modified RNA compared to unmodified RNA in the extremophilic bacterium Deinococcus radiodurans, which can endure high levels of oxidative stress. Two of the proteins, polynucleotide phosphorylase (PNPase) and DEAD-box RNA helicase (RhlB), interact with each other and reduce the cellular availability of 8-oxoG-modified RNA under oxidative stress. As such, this work contributes to our understanding of how RNA oxidation is influenced by RNA binding proteins in bacteria.

The challenges in finding your home as a multidisciplinary scientist.

Technological advances in a variety of scientific disciplines are being applied in the life sciences leading to an increase in the number scientists who see themselves or are classed as being multidisciplinary. Although their diverse skills are celebrated and needed to understand the immense complexity of life, being a multidisciplinary researcher can pose unique challenges. We asked multidisciplinary researchers and the director of an institute that fosters multidisciplinary research for their thoughts on what they see as the challenges or obstacles that multidisciplinary scientists can often face.

A strategy to quantify myofibroblast activation on a continuous spectrum.

Myofibroblasts are a highly secretory and contractile cell phenotype that are predominant in wound healing and fibrotic disease. Traditionally, myofibroblasts are identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers, leading to a binary classification: "activated" or "quiescent (non-activated)". More recently, however, myofibroblast activation has been considered on a continuous spectrum, but there is no established method to quantify the position of a cell on this spectrum. To this end, we developed a strategy based on microscopy imaging and machine learning methods to quantify myofibroblast activation in vitro on a continuous scale. We first measured morphological features of over 1000 individual cardiac fibroblasts and found that these features provide sufficient information to predict activation state. We next used dimensionality reduction techniques and self-supervised machine learning to create a continuous scale of activation based on features extracted from microscopy images. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of myofibroblast activation and provide an imaging-based strategy to quantify the position of a cell on that spectrum.

RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.

Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.

Engineering Toehold-Mediated Switches for Native RNA Detection and Regulation in Bacteria.

RNA switches are versatile tools in synthetic biology for sensing and regulation applications. The discoveries of RNA-mediated translational and transcriptional control have facilitated the development of complex de novo designs of RNA switches. Specifically, RNA toehold-mediated switches, in which binding to the toehold sensing domain controls the transition between switch states via strand displacement, have been extensively adapted for coupling systems responses to specific trans-RNA inputs. This review highlights some of the challenges associated with applying these switches for native RNA detection in vivo, including transferability between organisms. The applicability and design considerations of toehold-mediated switches are discussed by highlighting twelve recently developed switch designs. This review finishes with future perspectives to address current gaps in the field, particularly regarding the power of structural prediction algorithms for improved in vivo functionality of RNA switches.

Gestores de referencias bibliográficas y su impacto en las investigaciones / Bibliographic reference managers and their impact on research

Resumen La incorporación de los gestores de referencias bibliográficas a las investigaciones se debe a sus beneficios como organizador de citas, referencias y como un recurso informático para el desarrollo de proyectos. El objetivo de este artículo es actualizar conocimientos en los últimos 5 años, sobre el uso e implicación de los Gestores de Referencias en las investigaciones. Se realizó una revisión documental que permitió consultar 133 fuentes de información asociadas a cinco grupos de documentos: I. Rol que desempeñan los gestores de referencias bibliográficas en el desarrollo de habilidades informacionales y competencias investigativas. II. Uso de los gestores como herramientas en la investigación. III. Los gestores en temas de materiales didácticos. IV. En la confección de citas y referencias y V. Para otros objetivos de investigación. Se utilizaron el Zotero, el Excel 2013 y el paquete estadístico SPSS. Se calcularon frecuencias absolutas y porcentajes, así como la Moda para los grupos conformados. Entre otras conclusiones importantes, al analizar el comportamiento de la producción científica, la Moda señala al grupo II Uso de los gestores como herramientas en la investigación, como el más frecuente dentro de los documentos estudiados, por lo que se recomienda su estudio y actualización permanente.

Abstract The incorporation of bibliographic reference managers in research is due to its benefits as an organizer of citations, references and as an informatics resource for the development of projects. The objective of this article is to update knowledge in the last 5 years on the use and implication of reference managers in research. A documentary review was carried out which allowed to consult 133 sources of information associated with five groups of documents: I. Role of bibliographic reference managers in the development of informational skills and research competencies. II. Use of managers as tools in research. III. Managers in didactic materials issues. IV. In the preparation of citations and references and V. For other research objectives. Zotero, Excel 2013 and the SPSS statistical package were used. Absolute frequencies and percentages were calculated, as well as the mode for the groups formed. Among other important conclusions, when analyzing the behavior of scientific production, Moda points out group II Use of managers as tools in research, as the most frequent within the documents studied, so its study and permanent updating is recommended.

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) hosts an annual meeting series focused on presenting the latest research achievements involving RNA-based therapeutics and strategies, aiming to expand their current biomedical applications while overcoming the remaining challenges of the burgeoning field of RNA nanotechnology. The most recent online meeting hosted a series of engaging talks and discussions from an international cohort of leading nanotechnologists that focused on RNA modifications and modulation, dynamic RNA structures, overcoming delivery limitations using a variety of innovative platforms and approaches, and addressing the newly explored potential for immunomodulation with programmable nucleic acid nanoparticles. In this Nano Focus, we summarize the main discussion points, conclusions, and future directions identified during this two-day webinar as well as more recent advances to highlight and to accelerate this exciting field.

Small RNAs as a New Platform for Tuning the Biosynthesis of Silver Nanoparticles for Enhanced Material and Functional Properties.

Genetic engineering of nanoparticle biosynthesis in bacteria could help facilitate the production of nanoparticles with enhanced or desired properties. However, this process remains limited due to the lack of mechanistic knowledge regarding specific enzymes and other key biological factors. Herein, we report on the ability of small noncoding RNAs (sRNAs) to affect silver nanoparticle (AgNP) biosynthesis using the supernatant from the bacterium Deinococcus radiodurans. Deletion strains of 12 sRNAs potentially involved in the oxidative stress response were constructed, and the supernatants from these strains were screened for their effect on AgNP biosynthesis. We identified several sRNA deletions that drastically decreased AgNP yield compared to the wild-type (WT) strain, suggesting the importance of these sRNAs in AgNP biosynthesis. Furthermore, AgNPs biosynthesized using the supernatants from three of these sRNA deletion strains demonstrated significantly enhanced antimicrobial and catalytic activities against environmentally relevant dyes and bacteria relative to AgNPs biosynthesized using the WT strain. Characterization of these AgNPs using electron microscopy (EM), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) revealed that the deletion of these small RNAs led to changes within the supernatant composition that altered AgNP properties such as the surface chemistry, surface potential, and overall composition. Taken together, our results demonstrate that modulating specific sRNA levels can affect the composition of supernatants used to biosynthesize AgNPs, resulting in AgNPs with unique material properties and improved functionality; as such, we introduce sRNAs as a new platform for genetically engineering the biosynthesis of metal nanoparticles using bacteria. Many of the sRNAs examined in this work have potential regulatory roles in oxidative stress responses; further studies into their targets could help provide insight into the specific molecular mechanisms underlying bacterial biosynthesis and metal reduction, enabling the production of nanoparticles with enhanced properties.

Uncovering Transcriptional Regulators and Targets of sRNAs Using an Integrative Data-Mining Approach: H-NS-Regulated RseX as a Case Study.

Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo. We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection.

A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation.

Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance.