Faculty Bibliography

In the era of genome engineering, a new study returns to classical genetics to decipher genotype-phenotype relationships in unprecedented throughput and with unprecedented accuracy. Capitalizing on natural variation in yeast strains and frequent meiotic recombination, She and Jarosz (2018) dissect and map to nucleotide resolution, simple and complex determinants of diverse phenotypic traits.

DNA harbors the blueprint for life. However, the instructions stored in the DNA could be altered at the RNA level before they are executed. One of these processes is RNA editing, which was shown to modify RNA sequences in many organisms. The most abundant modification is the deamination of adenosine (A) into inosine (I). In turn, inosine can be identified as a guanosine (G) by the ribosome and other cellular machineries such as reverse transcriptase. In multicellular organisms, enzymes from the ADAR (adenosine deaminase acting on RNA) family mediate RNA editing in mRNA, whereas enzymes from the ADAT family mediate A-to-I editing on tRNAs. In bacteria however, until recently, only one editing site was described, in tRNA^Arg, but never in mRNA. The tRNA site was shown to be modified by tadA (tRNA specific adenosine deaminase) which is believed to be the ancestral enzyme for the RNA editing family of enzymes. In our recent work, we have shown for the first time, editing on multiple sites in bacterial mRNAs and identified tadA as the enzyme responsible for this editing activity. Focusing on one of the identified targets - the self-killing toxin hokB, we found that editing is physiologically regulated and that it increases protein activity. Here we discuss possible modes of regulation on hokB editing, potential roles of RNA editing in bacteria, possible implications, and future research directions.

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 Escherichia coli, 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 hokB 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 hokB 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.

Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems.

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

BACKGROUND:Cells constantly adapt to changes in their environment. When environment shifts between conditions that were previously encountered during the course of evolution, evolutionary-programmed responses are possible. Cells, however, may also encounter a new environment to which a novel response is required. To characterize the first steps in adaptation to a novel condition, we studied budding yeast growth on xylulose, a sugar that is very rarely found in the wild. RESULTS:We previously reported that growth on xylulose induces the expression of amino acid biosynthesis genes in multiple natural yeast isolates. This induction occurs despite the presence of amino acids in the growth medium and is a unique response to xylulose, not triggered by naturally available carbon sources. Propagating these strains for ~300 generations on xylulose significantly improved their growth rate. Notably, the most significant change in gene expression was the loss of amino acid biosynthesis gene induction. Furthermore, the reduction in amino-acid biosynthesis gene expression on xylulose was tightly correlated with the improvement in growth rate, suggesting that internal depletion of amino-acids presented a major bottleneck limiting growth in xylulose. CONCLUSIONS:We discuss the possible implications of our results for explaining how cells maintain the balance between supply and demand of amino acids during growth in evolutionary 'familiar' vs. 'novel' conditions.

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species.

Correlation does not imply causation. If two variables, say A and B, are correlated, it could be because A causes B, or that B causes A, or because a third factor affects them both. We suggest that in many cases in biology, the causal link might be bi-directional: A causes B through a fast-acting physiological process, while B causes A through a slowly accumulating evolutionary process. Furthermore, many trained biologists tend to consistently focus at first on the fast-acting direction, and overlook the slower process in the opposite direction. We analyse several examples from modern biology that demonstrate this bias (codon usage optimality and gene expression, gene duplication and genetic dispensability, stem cell division and cancer risk, and the microbiome and host metabolism) and also discuss an example from linguistics. These examples demonstrate mutual effects between the fast physiological processes and the slow evolutionary ones. We believe that building awareness of inference biases among biologists who tend to prefer one causal direction over another could improve scientific reasoning.