MinD-RNase E interplay controls localization of polar mRNAs in E. coli.

The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.

Spatiotemporal Organization of the E. coli Transcriptome: Translation Independence and Engagement in Regulation.

RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

Methods for studying RNA localization in bacteria.

The subcellular localization of RNA transcripts provides important insights into biological processes. Hence, understanding the mechanisms underlying RNA targeting is a high priority aim of modern cell biology. The advancements in imaging techniques, such as in situ hybridization and live-cell imaging, coupled with the evolution in optical microscopy led to the discovery that bacterial RNAs, despite the lack of nucleus, are specifically localized. Here we describe the methods used to study RNA localization in bacteria and their applications and discuss their advantages and limitations.

Protein targeting via mRNA in bacteria.

Proteins of all living organisms must reach their subcellular destination to sustain the cell structure and function. The proteins are transported to one of the cellular compartments, inserted into the membrane, or secreted across the membrane to the extracellular milieu. Cells have developed various mechanisms to transport proteins across membranes, among them localized translation. Evidence for targeting of Messenger RNA for the sake of translation of their respective protein products at specific subcellular sites in many eukaryotic model organisms have been accumulating in recent years. Cis-acting RNA localizing elements, termed RNA zip-codes, which are embedded within the mRNA sequence, are recognized by RNA-binding proteins, which in turn interact with motor proteins, thus coordinating the intracellular transport of the mRNA transcripts. Despite the rareness of conventional organelles, first and foremost a nucleus, pieces of evidence for mRNA localization to specific subcellular domains, where their protein products function, have also been obtained for prokaryotes. Although the underlying mechanisms for transcript localization in bacteria are yet to be unraveled, it is now obvious that intracellular localization of mRNA is a common mechanism to spatially localize proteins in both eukaryotes and prokaryotes. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

RNA localization in bacteria.

One of the most important discoveries in the field of microbiology in the last two decades is that bacterial cells have intricate subcellular organization. This understanding has emerged mainly from the depiction of spatial and temporal organization of proteins in specific domains within bacterial cells, e.g., midcell, cell poles, membrane and periplasm. Because translation of bacterial RNA molecules was considered to be strictly coupled to their synthesis, they were not thought to specifically localize to regions outside the nucleoid. However, the increasing interest in RNAs, including non-coding RNAs, encouraged researchers to explore the spatial and temporal localization of RNAs in bacteria. The recent technological improvements in the field of fluorescence microscopy allowed subcellular imaging of RNAs even in the tiny bacterial cells. It has been reported by several groups, including ours that transcripts may specifically localize in such cells. Here we review what is known about localization of RNA and of the pathways that determine RNA fate in bacteria, and discuss the possible cues and mechanisms underlying these distribution patterns.