Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Anionic G•U pairs in bacterial ribosomal rRNAs.

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G + 3 in the mRNA. In such pairs, the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.

Eukaryotic mRNAs contain a 5' cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

Regulating the ribosome: a spotlight on RNA dark matter.

In this issue, Pircher et al. (2014) show that an abundant ribosome-associated 18 nt noncoding RNA (ncRNA), derived from the open reading frame of an mRNA, acts directly on the ribosome and regulates global translation levels in response to hypertonic shock.

Cellular response to small molecules that selectively stall protein synthesis by the ribosome.

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.

PTBP1 mRNA isoforms and regulation of their translation.

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells and identified alternative 5' and 3' untranslated regions (5'-UTRs, 3'-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.

Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.].

eIF3 targets cell-proliferation messenger RNAs for translational activation or repression.

Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis and stress responses. The 13-subunit, 800-kilodalton eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). eIF3 binds to a highly specific program of messenger RNAs involved in cell growth control processes, including cell cycling, differentiation and apoptosis, via the mRNA 5' untranslated region. Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding the cell proliferation regulators c-JUN and BTG1 reveals that eIF3 uses different modes of RNA stem-loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis.

Differences in the path to exit the ribosome across the three domains of life.

The ribosome exit tunnel is an important structure involved in the regulation of translation and other essential functions such as protein folding. By comparing 20 recently obtained cryo-EM and X-ray crystallography structures of the ribosome from all three domains of life, we here characterize the key similarities and differences of the tunnel across species. We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure and sequence. We find that the tunnel is more conserved in the upper part close to the polypeptide transferase center, while in the lower part, it is substantially narrower in eukaryotes than in bacteria. Furthermore, we provide evidence for the existence of a second constriction site in eukaryotic exit tunnels. Overall, these results have several evolutionary and functional implications, which explain certain differences between eukaryotes and prokaryotes in their translation mechanisms. In particular, they suggest that major co-translational functions of bacterial tunnels were externalized in eukaryotes, while reducing the tunnel size provided some other advantages, such as facilitating the nascent chain elongation and enabling antibiotic resistance.

Programmable RNA recognition using a CRISPR-associated Argonaute.

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine-edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

Initiation of Protein Synthesis with Non-Canonical Amino Acids In†Vivo.

By transplanting identity elements into E. coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

Defects in the Assembly of Ribosomes Selected for ß-Amino Acid Incorporation.

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and ß-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo ß-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.

Quantitative determination of ribosome nascent chain stability.

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.