Improved RNA stability estimation indicates that transcriptional interference is frequent in diverse bacteria.

We used stochastic simulations and experimental data from E. coli, K. aerogenes, Synechococcus PCC 7002 and Synechocystis PCC 6803 to provide evidence that transcriptional interference via the collision mechanism is likely a prevalent mechanism for bacterial gene regulation. Rifampicin time-series data can be used to globally monitor and quantify collision between sense and antisense transcription-complexes. Our findings also highlight that transcriptional events, such as differential RNA decay, partial termination, and internal transcriptional start sites often deviate from gene annotations. Consequently, within a single gene annotation, there exist transcript segments with varying half-lives and transcriptional properties. To address these complexities, we introduce 'rifi', an R-package that analyzes transcriptomic data from rifampicin time series. 'rifi' employs a dynamic programming-based segmentation approach to identify individual transcripts, enabling accurate assessment of RNA stability and detection of diverse transcriptional events.

New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC.

Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in Bacillus subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in management of NAD+/NADH ratio in Firmicutes. IMPORTANCE Non-coding RNAs (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identifying the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify such targets and obtain the most complete list of targets for a defined sRNA.

Identification of an RNA sponge that controls the RoxS riboregulator of central metabolism in Bacillus subtilis.

sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.

Resequencing of a mutant bearing an iron starvation recovery phenotype defines Slr1658 as a new player in the regulatory network of a model cyanobacterium.

Photosynthetic microorganisms encounter an erratic nutrient environment characterized by periods of iron limitation and sufficiency. Surviving in such an environment requires mechanisms for handling these transitions. Our study identified a regulatory system involved in the process of recovery from iron limitation in cyanobacteria. We set out to study the role of bacterioferritin co-migratory proteins during transitions in iron bioavailability in the cyanobacterium Synechocystis sp. PCC 6803 using knockout strains coupled with physiological and biochemical measurements. One of the mutants displayed slow recovery from iron limitation. However, we discovered that the cause of the phenotype was not the intended knockout but rather the serendipitous selection of a mutation in an unrelated locus, slr1658. Bioinformatics analysis suggested similarities to two-component systems and a possible regulatory role. Transcriptomic analysis of the recovery from iron limitation showed that the slr1658 mutation had an extensive effect on the expression of genes encoding regulatory proteins, proteins involved in the remodeling and degradation of the photosynthetic apparatus and proteins modulating electron transport. Most significantly, expression of the cyanobacterial homologue of the cyclic electron transport protein PGR5 was upregulated 1000-fold in slr1658 disruption mutants. pgr5 transcripts in the Δslr1658 mutant retained these high levels under a range of stress and recovery conditions. The results suggest that slr1658 is part of a regulatory operon that, among other aspects, affects the regulation of alternative electron flow. Disruption of its function has deleterious results under oxidative stress promoting conditions.

Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the sRNA IsaR1.

Oxygenic photosynthesis crucially depends on proteins that possess Fe2+ or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe2+-containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms.

Computational selection and experimental validation of allosteric ribozymes that sense a specific sequence of human telomerase reverse transcriptase mRNAs as universal anticancer therapy agents.

High expression levels of telomerase reverse transcriptase messenger RNAs in differentiated cells can be used as a common marker for cancer development. In this paper, we describe a novel computational method for selection of allosteric ribozymes that sense a specific sequence of human telomerase reverse transcriptase mRNAs. The in silico selection employed is based on computing secondary structures of RNA using the partition function in combination with a random search algorithm. We selected one of the ribozymes for experimental validation. The obtained results demonstrate that the tested ribozyme has a high-speed (∼1.8 per minute) of self-cleavage and is very selective. It can distinguish well between perfectly matching effector and the closest expressed RNA sequence in the human cell with 10 mismatches, with a ∼300-fold difference under physiologically relevant conditions. The presented algorithm is universal since the allosteric ribozymes can be designed to sense any specific RNA or DNA sequence of interest. Such designer ribozymes may be used for monitoring the expression of mRNAs in the cell and for developing novel anticancer gene therapies.