RNA Localization in Bacteria

Abstract

Diverse mechanisms and functions of posttranscriptional regulation by small regulatory RNAs and RNA-binding proteins 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 to the small size of bacterial cells, RNA localization and localization-associated functions 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 gene expression and 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.

Figure 1:. Methods to visualize bacterial RNAs.

(A) smFISH and its application to image SgrS and its target mRNA ptsG (adapted from (105)). Images from diffraction-limited and super-resolution microscopes are shown for comparison. (B) Illustration of the fluorescent protein RBP-RNA aptamer approach, using the MS2 system as an example, and its application to track mRNAs at the single-molecule level in live E.coli cells (image adapted from (67)). The scale bar in the image represents 2 μm. (C) The Spinach aptamer and its application to image mRNAs in live E.coli cells. The image is adapted from (58) (licensed under a Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/), in which a tandem array of Spinach aptamers is fused to the RNA of interest to enhance the signal, and fluorescent detection upon addition of the organic ligand DFHBI.

Figure 2:. Diverse patterns of subcellular mRNA localization in bacteria.

Schematic drawings of diverse mRNA localization patterns commonly reported in different bacteria. RNA molecules are shown in green, and the nucleoid in grey. (A) Distribution throughout the cytoplasm. (B) Localization at the site of transcription in the nucleoid. (C) Helical localization. (D) Enrichment at the inner membrane. (E) Localization at the cell poles and (F) septum.

Figure 3:. Emerging mRNA imaging methods in eukaryotic systems.

(A) In the in situ hybridization chain reaction (HCR), binding of the primary probe initiates the alternating binding of two HCR probes thereby amplifying the signal. (B) In the in situ polymerase chain reaction (PCR), a cDNA is first generated from the RNA of interest. Padlock probes are hybridized to the cDNA and ligated to be circular DNAs. Fluorophore labeled secondary probes are then hybridized to the products generated from rolling circle amplification of these circular DNA templates. (C) Schematic representation of the TRICK reporter construct. (D) Schematic representation of the SunTag construct. (E) Schematic representation of the TREAT reporter construct.