Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing

Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability

Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications.

The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNA(Leu)(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation

Conservative route to genome compaction in a miniature annelid

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5′ ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of rules of transcription initiation, led to discovery of new core promoter sequence features and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 micrograms), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5′ ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods by generating a complex, high quality library from mouse embryonic day (E) 11.5 primordial germ cells

Neuronal genes deregulated in Cornelia de Lange Syndrome respond to removal and reexpression of cohesin

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS

DNA stretching induces Cas9 off-target activity

CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms

DNA stretching induces Cas9 off-target binding and cleavage

CRISPR/Cas9 is a powerful genome editing tool, but spurious off-target edits present a barrier towards therapeutic applications. To understand how CRISPR/Cas9 discrimi-nates between on- and off-targets, we have developed a single-molecule assay com-bining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule FRET and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with 10 mis-matches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (e.g., transcription, replication, etc) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms

On the Mechanism and Origin of Isoleucyl-tRNA Synthetase Editing against Norvaline.

Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins

Publisher Correction: Conservative route to genome compaction in a miniature annelid.

A Correction to this paper has been published: https://doi.org/10.1038/s41559-020-01327-6

Conservative route to genome compaction in a miniature annelid

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.This study was supported by Sars Centre core budget and the European Research Council (ERC) grant agreement no. 648861 to A.H. J.M.M.-D. was additionally supported by the ERC grant agreement no. 801669, and B.C.V. by an EMBO Long-Term Fellowship (ALTF 74-2018). J.L.G.-S. received funding from the ERC (grant agreement no. 740041) and the Spanish Ministerio de Economía y Competitividad (grant no. BFU2016-74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687