Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control.

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

Coral Reef Population Genomics in an Age of Global Change.

Coral reefs are both exceptionally biodiverse and threatened by climate change and other human activities. Here, we review population genomic processes in coral reef taxa and their importance for understanding responses to global change. Many taxa on coral reefs are characterized by weak genetic drift, extensive gene flow, and strong selection from complex biotic and abiotic environments, which together present a fascinating test of microevolutionary theory. Selection, gene flow, and hybridization have played and will continue to play an important role in the adaptation or extinction of coral reef taxa in the face of rapid environmental change, but research remains exceptionally limited compared to the urgent needs. Critical areas for future investigation include understanding evolutionary potential and the mechanisms of local adaptation, developing historical baselines, and building greater research capacity in the countries where most reef diversity is concentrated.

Ribosome quality control antagonizes the activation of the integrated stress response on colliding ribosomes.

Stalling during translation triggers ribosome quality control (RQC) to maintain proteostasis. Recently, stalling has also been linked to the activation of integrated stress response (ISR) by Gcn2. How the two processes are coordinated is unclear. Here, we show that activation of RQC by Hel2 suppresses that of Gcn2. We further show that Hel2 and Gcn2 are activated by a similar set of agents that cause ribosome stalling, with maximal activation of Hel2 observed at a lower frequency of stalling. Interestingly, inactivation of one pathway was found to result in the overactivation of the other, suggesting that both are activated by the same signal of ribosome collisions. Notably, the processes do not appear to be in direct competition with each other; ISR prefers a vacant A site, whereas RQC displays no preference. Collectively, our findings provide important details about how multiple pathways that recognize stalled ribosomes coordinate to mount the appropriate response.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Increasing environmental variability inhibits evolutionary rescue in a long-lived vertebrate.

Evolutionary rescue, whereby adaptive evolutionary change rescues populations from extinction, is theorized to enable imperiled animal populations to persist under increasing anthropogenic change. Despite a large body of evidence in theoretical and laboratory settings, the potential for evolutionary rescue to be a viable adaptation process for free-ranging animals remains unknown. Here, we leverage a 38-year dataset following the fates of 53,959 Magellanic penguins (Spheniscus magellanicus) to investigate whether a free-ranging vertebrate species can morphologically adapt to long-term environmental change sufficiently to promote population persistence. Despite strong selective pressures, we found that penguins did not adapt morphologically to long-term environmental changes, leading to projected population extirpation. Fluctuating selection benefited larger penguins in some environmental contexts, and smaller penguins in others, ultimately mitigating their ability to adapt under increasing environmental variability. Under future climate projections, we found that the species cannot be rescued by adaptation, suggesting similar constraints for other long-lived species. Such results reveal how fluctuating selection driven by environmental variability can inhibit adaptation under long-term environmental change. Our eco-evolutionary approach helps explain the lack of adaptation and evolutionary rescue in response to environmental change observed in many animal species.

Prepping for climate change by introgressive hybridization.

Species and populations may adapt to climate change by microevolutionary processes. However, standing genetic variation can be insufficient for this to occur. An interesting new study of a system of rainbowfish species shows that intraspecific hybridization enriches gene pools with adaptive variation that may allow persistence in a changing climate.

Whole Genomes Inform Genetic Rescue Strategy for Montane Red Foxes in North America.

A few iconic examples have proven the value of facilitated gene flow for counteracting inbreeding depression and staving off extinction; yet, the practice is often not implemented for fear of causing outbreeding depression. Using genomic sequencing, climatic niche modeling, and demographic reconstruction, we sought to assess the risks and benefits of using translocations as a tool for recovery of endangered montane red fox (Vulpes vulpes) populations in the western United States. We demonstrated elevated inbreeding and homozygosity of deleterious alleles across all populations, but especially those isolated in the Cascade and Sierra Nevada ranges. Consequently, translocations would be expected to increase population growth by masking deleterious recessive alleles. Demographic reconstructions further indicated shallow divergences of less than a few thousand years among montane populations, suggesting low risk of outbreeding depression. These genomic-guided findings set the stage for future management, the documentation of which will provide a roadmap for recovery of other data-deficient taxa.

Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids.

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB.

RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.

A theory of resistance to multiplexed gene drive demonstrates the significant role of weakly deleterious natural genetic variation.

Evolution of resistance is a major barrier to successful deployment of gene-drive systems to suppress natural populations, which could greatly reduce the burden of many vector-borne diseases. Multiplexed guide RNAs (gRNAs) that require resistance mutations in all target cut sites are a promising antiresistance strategy since, in principle, resistance would only arise in unrealistically large populations. Using stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) nonhomologous end-joining mutations, 2) single-nucleotide mutants arising de novo, or 3) single-nucleotide polymorphisms preexisting as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation-selection-drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find that this leads to a phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multisite resistance compared to de novo mutation. This key result provides design criterion for antiresistance multiplexed systems, which, in general, will need a larger number of gRNAs compared to de novo expectations. This theory may have wider application to the evolution of resistance or evolutionary rescue when multiple changes are required before selection can act.

Evolution of plasticity prevents postinvasion extinction of a native forb.

Exotic plant invaders pose a serious threat to native plants. However, despite showing inferior competitive ability and decreased performance, native species often subsist in invaded communities. The decline of native populations is hypothesized to be halted and eventually reversed if adaptive evolutionary changes can keep up with the environmental stress induced by invaders, that is, when population extinction is prevented by evolutionary rescue (ER). Nevertheless, evidence for the role of ER in postinvasion persistence of native flora remains scarce. Here, I explored the population density of a native forb, Veronica chamaedrys, and evaluated the changes in the shade-responsive traits of its populations distributed along the invasion chronosequence of an exotic transformer, Heracleum mantegazzianum, which was replicated in five areas. I found a U-shaped population trajectory that paralleled the evolution of plasticity to shade. Whereas V. chamaedrys genotypes from intact, more open sites exhibited a shade-tolerance strategy (pronounced leaf area/mass ratio), reduced light availability at the invaded sites selected for a shade-avoidance strategy (greater internode elongation). Field experiments subsequently confirmed that the shifts in shade-response strategies were adaptive and secured postinvasion population persistence, as indicated by further modeling. Alternative ecological mechanisms (habitat improvement or arrival of immigrants) were less likely explanations than ER for the observed population rebound, although the contribution of maternal effects cannot be dismissed. These results suggest that V. chamaedrys survived because of adaptive evolutionary changes operating on the same timescale as the invasion-induced stress, but the generality of ER for postinvasion persistence of native plants remains unknown.

The MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4) is essential for the accumulation of dicistronic rpl5-cob mRNAs in Arabidopsis thaliana.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3' termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3' end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.

Behavioural plasticity compensates for adaptive loss of cricket song.

Behavioural flexibility might help animals cope with costs of genetic variants under selection, promoting genetic adaptation. However, it has proven challenging to experimentally link behavioural flexibility to the predicted compensation of population-level fitness. We tested this prediction using the field cricket Teleogryllus oceanicus. In Hawaiian populations, a mutation silences males and protects against eavesdropping parasitoids. To examine how the loss of this critical acoustic communication signal impacts offspring production and mate location, we developed a high-resolution, individual-based tracking system for low-light, naturalistic conditions. Offspring production did not differ significantly in replicate silent versus singing populations, and fitness compensation in silent conditions was associated with significantly increased locomotion in both sexes. Our results provide evidence that flexible behaviour can promote genetic adaptation via compensation in reproductive output and suggest that rapid evolution of animal communication systems may be less constrained than previously appreciated.

Environmental complexity mitigates the demographic impact of sexual selection.

Sexual selection and the evolution of costly mating strategies can negatively impact population viability and adaptive potential. While laboratory studies have documented outcomes stemming from these processes, recent observations suggest that the demographic impact of sexual selection is contingent on the environment and therefore may have been overestimated in simple laboratory settings. Here we find support for this claim. We exposed copies of beetle populations, previously evolved with or without sexual selection, to a 10-generation heatwave while maintaining half of them in a simple environment and the other half in a complex environment. Populations with an evolutionary history of sexual selection maintained larger sizes and more stable growth rates in complex (relative to simple) environments, an effect not seen in populations evolved without sexual selection. These results have implications for evolutionary forecasting and suggest that the negative demographic impact of sexually selected mating strategies might be low in natural populations.

Genetic Incompatibilities and Evolutionary Rescue by Wild Relatives Shaped Grain Amaranth Domestication.

Crop domestication and the subsequent expansion of crops have long been thought of as a linear process from a wild ancestor to a domesticate. However, evidence of gene flow from locally adapted wild relatives that provided adaptive alleles into crops has been identified in multiple species. Yet, little is known about the evolutionary consequences of gene flow during domestication and the interaction of gene flow and genetic load in crop populations. We study the pseudo-cereal grain amaranth that has been domesticated three times in different geographic regions of the Americas. We quantify the amount and distribution of gene flow and genetic load along the genome of the three grain amaranth species and their two wild relatives. Our results show ample gene flow between crop species and between crops and their wild relatives. Gene flow from wild relatives decreased genetic load in the three crop species. This suggests that wild relatives could provide evolutionary rescue by replacing deleterious alleles in crops. We assess experimental hybrids between the three crop species and found genetic incompatibilities between one Central American grain amaranth and the other two crop species. These incompatibilities might have created recent reproductive barriers and maintained species integrity today. Together, our results show that gene flow played an important role in the domestication and expansion of grain amaranth, despite genetic species barriers. The domestication of plants was likely not linear and created a genomic mosaic by multiple contributors with varying fitness effects for today's crops.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

Prophage excision switches the primary ribosome rescue pathway and rescue-associated gene regulations in Escherichia coli.

Escherichia coli has multiple pathways to release nonproductive ribosome complexes stalled at the 3' end of nonstop mRNA: tmRNA (SsrA RNA)-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4-57 prophage adjacent to E. coli ssrA leads to the inactivation of tmRNA and switches the primary rescue pathway from trans-translation to ArfA/RF2. This "rescue-switching" rearranges not only the proteome landscape in E. coli but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA+ cells, we found ZntR, whose mRNA is transcribed together as the upstream part of nonstop arfA mRNA. Repression of ZntR and reconstituted model genes depends on the translation of the downstream nonstop ORFs that trigger the trans-translation-coupled exonucleolytic degradation by polynucleotide phosphorylase (PNPase). Namely, our studies provide a novel example of trans-translation-dependent regulation and re-define the physiological roles of prophage excision.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Programmable C-to-U RNA editing using the human APOBEC3A deaminase.

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.

Long-term assessment of haematological recovery following somatic genetic rescue in a MYSM1-deficient patient: Implications for in vivo gene therapy.

MYSM1 deficiency causes inherited bone marrow failure syndrome (IBMFS). We have previously identified an IBMFS patient with a homozygous pathogenic variant in MYSM1 who recovered from cytopenia due to spontaneous correction of one MYSM1 variant in the haematopoietic compartment, an event called somatic genetic rescue (SGR). The study of the genetic and biological aspects of the patient's haematopoietic/lymphopoietic system over a decade after SGR shows that one genetically corrected haematopoietic stem cell (HSC) can restore a healthy and stable haematopoietic system. This supports in vivo gene correction of HSCs as a promising treatment for IBMFS, including MYSM1 deficiency.