Nucleoid and cytoplasmic localization of small RNAs inEscherichia coli

Abstract

Bacterial small RNAs (sRNAs) regulate protein production by binding to mRNAs and altering their translation and degradation. sRNAs are smaller than most mRNAs but larger than many proteins. Therefore it is uncertain whether sRNAs can enter the nucleoid to target nascent mRNAs. Here, we investigate the intracellular localization of sRNAs transcribed from plasmids in Escherichia coli using RNA fluorescent in-situ hybridization. We found that sRNAs (GlmZ, OxyS, RyhB and SgrS) have equal preference for the nucleoid and cytoplasm, and no preferential localization at the cell membrane. We show using the gfp mRNA (encoding green fluorescent protein) that non-sRNAs can be engineered to have different proportions of nucleoid and cytoplasmic localization by altering their length and/or translation. The same localization as sRNAs was achieved by decreasing gfp mRNA length and translation, which suggests that sRNAs and other RNAs may enter the densely packed DNA of the nucleoid if they are sufficiently small. We also found that the Hfq protein, which binds sRNAs, minimally affects sRNA localization. Important implications of our findings for engineering synthetic circuits are: (i) sRNAs can potentially bind nascent mRNAs in the nucleoid, and (ii) localization patterns and distribution volumes of sRNAs can differ from some larger RNAs.

RNA Localization in Bacteria

“…showed that SgrS, an sRNA involved in the glucose-phosphate stress response, is roughly homogeneously distributed in the cytoplasm when its copy number is high, whereas when its expression is lower, it appears to be specifically absent from the central nucleoid region (105). A more comprehensive study of the localization of several several sRNAs, including GlmZ, OxyS, RyhB and SgrS, found equal preference for localization within cytoplasm and nucleoid region and no preferential membrane localization, or cell poles, as often observed for mRNAs by quantifying the signal overlap between RNA FISH and DAPI staining of DNA (106). Interestingly, the ability of a particular transcript to freely diffuse into the nucleoid region was correlated with the length and the translation activity of the RNA, because shortening and reducing translation of gfp mRNA led to the same localization as sRNAs and reduced nucleoid exclusion.…”

“…Therefore, in general, longer RNAs, including coding RNAs, have a lower propensity towards nucleoid localization compared to shorter, often non-coding transcripts. So far, the available studies on a handful of examples of sRNAs suggest an unbiased cellular localization (105, 106). It should be noted that investigation of sRNA localizations are predominately focused on non-coding regulatory RNAs.…”

“…Future experiments are needed to resolve this discrepancy in observation of Hfq localization. Despite absence of Hfq significantly affecting the stability and abundance of sRNAs (105), it has minimal effect on the localization of tested sRNAs (106). As mentioned above, localization of the ptsG mRNA to the inner membrane has been reported to strongly contribute to its efficient repression by the sRNA SgrS, together with Hfq, during phosphosugar stress (111).…”

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

“…Although a role for specific RNA binding proteins cannot be ruled out as drivers of the process, a simpler explanation is that the corresponding sequences endow the xyl transcripts with physical properties that make them to be quickly discharged from the vicinity of the nucleoid, especially if the transcribed sequences are large. In fact, it seems that smaller RNAs use to appear uniformly distributed throughout the bacterial cell, while longer molecules typically display more limited dispersion (47). Large mRNAs can hardly co-localize with densely packed DNA regions due to straight physical forces e.g.…”

“…However, should transcript diffusion away from the promoter and capture by ribosomes in a different cell location occur, confinement in given spots for translation could become apparent. Some mRNAs can indeed travel throughout the E. coli's interior (64) and translation inhibition makes RNA to remain closer to the nucleoid (47). This suggests that at least in some cases ribosomes in fact pull mRNA from the nucleoid towards the rest of the cytoplasm.…”