Faculty Bibliography

Fitness landscape mapping and the prediction of evolutionary trajectories on these landscapes are major tasks in evolutionary biology research. Evolutionary dynamics is tightly linked to the landscape topography, but this relation is not straightforward. Here, we analyze a fitness landscape of a yeast tRNA gene, previously measured under four different conditions. We find that the wild type allele is sub-optimal, and 8%-10% of its variants are fitter. We rule out the possibilities that the wild type is fittest on average on these four conditions or located on a local fitness maximum. Notwithstanding, we cannot exclude the possibility that the wild type might be fittest in some of the many conditions in the complex ecology that yeast lives at. Instead, we find that the wild type is mutationally robust ('flat'), while more fit variants are typically mutationally fragile. Similar observations of mutational robustness or flatness have been so far made in very few cases, predominantly in viral genomes.

The COVID-19 pandemic demonstrated that the process of global vaccination against a novel virus can be a prolonged one. Social distancing measures, that are initially adopted to control the pandemic, are gradually relaxed as vaccination progresses and population immunity increases. The result is a prolonged period of high disease prevalence combined with a fitness advantage for vaccine-resistant variants, which together lead to a considerably increased probability for vaccine escape. A spatial vaccination strategy is proposed that has the potential to dramatically reduce this risk. Rather than dispersing the vaccination effort evenly throughout a country, distinct geographic regions of the country are sequentially vaccinated, quickly bringing each to effective herd immunity. Regions with high vaccination rates will then have low infection rates and vice versa. Since people primarily interact within their own region, spatial vaccination reduces the number of encounters between infected individuals (the source of mutations) and vaccinated individuals (who facilitate the spread of vaccine-resistant strains). Thus, spatial vaccination may help mitigate the global risk of vaccine-resistant variants.

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2'-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.

The greatest hope for a return to normalcy following the COVID-19 pandemic is worldwide vaccination. Yet, a relaxation of social distancing that allows increased transmissibility, coupled with selection pressure due to vaccination, will probably lead to the emergence of vaccine resistance. We analyse the evolutionary dynamics of COVID-19 in the presence of dynamic contact reduction and in response to vaccination. We use infection and vaccination data from six different countries. We show that under slow vaccination, resistance is very likely to appear even if social distancing is maintained. Under fast vaccination, the emergence of mutants can be prevented if social distancing is maintained during vaccination. We analyse multiple human factors that affect the evolutionary potential of the virus, including the extent of dynamic social distancing, vaccination campaigns, vaccine design, boosters and vaccine hesitancy. We provide guidelines for policies that aim to minimize the probability of emergence of vaccine-resistant variants.

The tRNA pool determines the efficiency, throughput, and accuracy of translation. Previous studies have identified dynamic changes in the tRNA (transfer RNA) supply and mRNA (messenger RNA) demand during cancerous proliferation. Yet dynamic changes may also occur during physiologically normal proliferation, and these are less well characterized. We examined the tRNA and mRNA pools of T cells during their vigorous proliferation and differentiation upon triggering their antigen receptor. We observed a global signature of switch in demand for codons at the early proliferation phase of the response, accompanied by corresponding changes in tRNA expression levels. In the later phase, upon differentiation, the response of the tRNA pool relaxed back to the basal level, potentially restraining excessive proliferation. Sequencing of tRNAs allowed us to evaluate their diverse base-modifications. We found that two types of tRNA modifications, wybutosine and ms²t6A, are reduced dramatically during T cell activation. These modifications occur in the anticodon loops of two tRNAs that decode "slippery codons," which are prone to ribosomal frameshifting. Attenuation of these frameshift-protective modifications is expected to increase the potential for proteome-wide frameshifting during T cell proliferation. Indeed, human cell lines deleted of a wybutosine writer showed increased ribosomal frameshifting, as detected with an HIV gag-pol frameshifting site reporter. These results may explain HIV's specific tropism toward proliferating T cells since it requires ribosomal frameshift exactly on the corresponding codon for infection. The changes in tRNA expression and modifications uncover a layer of translation regulation during T cell proliferation and expose a potential tradeoff between cellular growth and translation fidelity.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations.

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

Different subsets of the tRNA pool in human cells are expressed in different cellular conditions. The 'proliferation-tRNAs' are induced upon normal and cancerous cell division, while the 'differentiation-tRNAs' are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation-tRNAs are essential compared to differentiation- tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation-tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.

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) vs. 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 hotspots. 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.