Adenosine (A) to inosine (I) RNA editing is widespread in eukaryotes. In prokaryotes, however, A-to-I RNA editing was only reported to occur in tRNAs but not in protein-coding genes. By comparing DNA and RNA sequences of , we show for the first time that A-to-I editing occurs also in prokaryotic mRNAs and has the potential to affect the translated proteins and cell physiology. We found 15 novel A-to-I editing events, of which 12 occurred within known protein-coding genes where they always recode a tyrosine (TAC) into a cysteine (TGC) codon. Furthermore, we identified the tRNA-specific adenosine deaminase (tadA) as the editing enzyme of all these editing sites, thus making it the first identified RNA editing enzyme that modifies both tRNAs and mRNAs. Interestingly, several of the editing targets are self-killing toxins that belong to evolutionarily conserved toxin-antitoxin pairs. We focused on hokB, a toxin that confers antibiotic tolerance by growth inhibition, as it demonstrated the highest level of such mRNA editing. We identified a correlated mutation pattern between the edited and a DNA hard-coded Cys residue positions in the toxin and demonstrated that RNA editing occurs in in two additional bacterial species. Thus, not only the toxin is evolutionarily conserved but also the editing itself within the toxin is. Finally, we found that RNA editing in increases as a function of cell density and enhances its toxicity. Our work thus demonstrates the occurrence, regulation, and functional consequences of RNA editing in bacteria.
Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3’UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3’UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3’UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3’UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3’UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.
Tracing evolutionary processes that lead to fixation of genomic variation in wild bacterial populations is a prime challenge in molecular evolution. In particular, the relative contribution of Horizontal Gene Transfer (HGT) versus de novo mutations during adaptation to a new environment is poorly understood. To gain a better understanding of the dynamics of HGT and its effect on adaptation, we subjected several populations of competent Bacillus subtilis to a serial dilution evolution on a high salt containing medium, either with or without foreign DNA from diverse pre-adapted or naturally salt tolerant species. Following 504 generations of evolution, all populations improved growth yield on the medium. Sequencing of evolved populations revealed extensive acquisition of foreign DNA from close Bacillus donors but not from more remote donors. HGT occurred in bursts, whereby a single bacterial cell appears to have acquired dozens of fragments at once. In the largest burst, close to 2% of the genome has been replaced by HGT. Acquired segments tend to be clustered in integration "hot spots". Other than HGT, genomes also acquired spontaneous mutations. Many of these mutations occurred within, and seem to alter, the sequence of flagellar proteins. Finally, we show that while some HGT fragments could be neutral, others are adaptive and accelerate evolution.
Target-oriented substructure-based virtual screening (sSBVS) of molecules is a promising approach in drug discovery. Yet, there are doubts whether sSBVS is suitable also for extrapolation, that is, for detecting molecules that are very different from those used for training. Herein, we evaluate the predictive power of classic virtual screening methods, namely, similarity searching using Tanimoto coefficient (MTC) and Naive Bayes (NB). As could be expected, these classic methods perform better in interpolation than in extrapolation tasks. Consequently, to enhance the predictive ability for extrapolation tasks, we introduce the Shadow approach, in which inclusion relations between substructures are considered, as opposed to the classic sSBVS methods that assume independence between substructures. Specifically, we discard contributions from substructures included in ("shaded" by) others which are, in turn, included in the molecule of interest. Indeed, the Shadow classifier significantly outperforms both MTC (pValue = 3.1 × 10(-16)) and NB (pValue = 3.5 × 10(-9)) in detecting hits sharing low similarity with the training active molecules.
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