The world of ribonucleases from pseudomonads: a short trip through the main features and singularities.

The development of synthetic biology has brought an unprecedented increase in the number molecular tools applicable into a microbial chassis. The exploration of such tools into different bacteria revealed not only the challenges of context dependency of biological functions but also the complexity and diversity of regulatory layers in bacterial cells. Most of the standardized genetic tools and principles/functions have been mostly based on model microorganisms, namely Escherichia coli. In contrast, the non-model pseudomonads lack a deeper understanding of their regulatory layers and have limited molecular tools. They are resistant pathogens and promising alternative bacterial chassis, making them attractive targets for further studies. Ribonucleases (RNases) are key players in the post-transcriptional control of gene expression by degrading or processing the RNA molecules in the cell. These enzymes act according to the cellular requirements and can also be seen as the recyclers of ribonucleotides, allowing a continuous input of these cellular resources. This makes these post-transcriptional regulators perfect candidates to regulate microbial physiology. This review summarizes the current knowledge and unique properties of ribonucleases in the world of pseudomonads, taking into account genomic context analysis, biological function and strategies to use ribonucleases to improve biotechnological processes.

Ribonucleases control distinct traits of Pseudomonas putida lifestyle.

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

Tailor-made sRNAs: a plasmid tool to control the expression of target mRNAs in Pseudomonas putida.

Pseudomonas putida is a highly attractive production system for industrial needs. However, for its improvement as a biocatalyst at the industrial level, modulation of its gene expression is urgently needed. We report the construction of a plasmid expressing a small RNA-based system with the potential to be used for different purposes. Due to the small RNAs modular composition, the design facilities and ability to tune gene expression, they constitute a powerful tool in genetic and metabolic engineering. In the tool presented here, customized sRNAs are expressed from a plasmid and specifically directed to any region of a chosen target. Expression of these customized sRNAs is shown to differentially modulate the level of endogenous and heterologous reporter genes. The antisense interaction of the sRNA with the mRNA produces different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we demonstrate the duality of this system, which is able either to decrease or increase the expression of the same given gene. This system combines high specificity with the potential to be widely applied, due to its predicted ability to modulate the expression of virtually any given gene. This plasmid can be used to redesign P. putida metabolism, fulfilling an important industrial gap.

Reprogramming bacteria with RNA regulators.

The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more 'economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.

Modulating Heterologous Gene Expression with Portable mRNA-Stabilizing 5'-UTR Sequences.

RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E. coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5'-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.

Surprises in the 3'-end: 'U' can decide too!

RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5'- or 3'- extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3'-end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3'-RNA modification, both in eukaryotes and prokaryotes. The importance of 3'-oligouridylation has recently gained attention through the discovery of several types of uridylated-RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3'-5' exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)-tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3'-terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3'-RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3'-end modification and the effectors that are selectively activated by this process.

The 3'-5' exoribonuclease Dis3 regulates the expression of specific microRNAs in Drosophila wing imaginal discs.

Dis3 is a highly conserved exoribonuclease which degrades RNAs in the 3'-5' direction. Mutations in Dis3 are associated with a number of human cancers including multiple myeloma and acute myeloid leukemia. In this work, we have assessed the effect of a Dis3 knockdown on Drosophila imaginal disc development and on expression of mature microRNAs. We find that Dis3 knockdown severely disrupts the development of wing imaginal discs in that the flies have a "no wing" phenotype. Use of RNA-seq to quantify the effect of Dis3 knockdown on microRNA expression shows that Dis3 normally regulates a small subset of microRNAs, with only 11 (10.1%) increasing in level ≥ 2-fold and 6 (5.5%) decreasing in level ≥ 2-fold. Of these microRNAs, miR-252-5p is increased 2.1-fold in Dis3-depleted cells compared to controls while the level of the miR-252 precursor is unchanged, suggesting that Dis3 can act in the cytoplasm to specifically degrade this mature miRNA. Furthermore, our experiments suggest that Dis3 normally interacts with the exosomal subunit Rrp40 in the cytoplasm to target miR-252-5p for degradation during normal wing development. Another microRNA, miR-982-5p, is expressed at lower levels in Dis3 knockdown cells, while the miR-982 precursor remains unchanged, indicating that Dis3 is involved in its processing. Our study therefore reveals an unexpected specificity for this ribonuclease toward microRNA regulation, which is likely to be conserved in other eukaryotes and may be relevant to understanding its role in human disease.