Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

Gijtenbeek, Lieke A van

doi:10.3389/fmicb.2017.01161

Cited by 21 publications

(18 citation statements)

References 182 publications

(304 reference statements)

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“…Here, we first introduce labeling and imaging methods used in bacterial systems (see also several recent reviews on RNA imaging methods (2, 17, 18)).…”

Section: Approaches To Study Rna Localizationmentioning

confidence: 99%

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

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Diverse mechanisms and functions of post-transcriptional regulation by small regulatory RNAs (sRNAs) and RNA binding proteins (RBPs) have been described in bacteria. In contrast, little is known about the spatial organization of RNAs in bacterial cells. In eukaryotes, subcellular localization and transport of RNAs play important roles in diverse physiological processes, such as embryonic patterning, asymmetric cell division, epithelial polarity, and neuronal plasticity. It is now clear that bacterial RNAs also can accumulate at distinct sites in the cell. However, due the small size of bacterial cells, localized RNA and translation are more challenging to study in bacterial cells, and the underlying molecular mechanisms of transcript localization are less understood. Here we review the emerging examples of RNAs localized to specific subcellular locations in bacteria, with indications that subcellular localization of transcripts might be important for regulatory processes. Diverse mechanisms for bacterial RNA localization have been suggested, including close association to their genomic site of transcription, or to the localizations of their protein products in translation-dependent or independent processes. We also provide an overview of the state-of-the-art of technologies to visualize and track bacterial RNAs, ranging from hybridization-based approaches in fixed cells to in vivo imaging approaches using fluorescent protein reporters and/or RNA aptamers in single living bacterial cells. We conclude with a discussion of open questions in the field and ongoing technological developments regarding RNA imaging in eukaryotic systems that might likewise provide novel insights into RNA localization in bacteria.

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“…Here, we first introduce labeling and imaging methods used in bacterial systems (see also several recent reviews on RNA imaging methods (2, 17, 18)).…”

Section: Approaches To Study Rna Localizationmentioning

confidence: 99%

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

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“…In contrast to DNA, there are not many RNA dyes available. Since research has mainly focused on visualizing specific transcripts, sequence-specific RNA labeling with RNA-binding proteins and hybridization techniques is well-established (van Gijtenbeek and Kok, 2017;Fei and Sharma, 2018). One general RNA dye is the green-fluorescent SYTO variant RNASelect, which is five times more selective for RNA over DNA and has been successfully employed for staining of RNA in live E. coli (Bakshi et al, 2014).…”

Section: Nucleic Acidsmentioning

confidence: 99%

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides

Schäfer

Wenzel

2020

Front. Cell. Infect. Microbiol.

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Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.

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“…Small hairpin RNA aptamers are naturally found in certain mRNAs [ 1 , 2 , 3 ] and other RNAs [ 4 ]. Diverse hairpin aptamers have been used for tagging many kinds of RNA molecules for recruiting their cognate target proteins or ligands on the fusion RNA molecules [ 4 , 5 , 6 , 7 ]. On the other hand, transfer RNAs (tRNAs) are small RNA molecules that transfer proteinogenic amino acids onto the growing polypeptide chain in the ribosome.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Aptamer-Tagged tRNAs

Mukai

2020

IJMS

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Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

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Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

Cited by 21 publications

References 182 publications

RNA Localization in Bacteria

RNA Localization in Bacteria

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides

Rational Design of Aptamer-Tagged tRNAs

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