Biology subjects

Niwa, T.

Publications and source records attributed to Niwa, T..

At least 19 recordsLinked to original sources

bioRxiv2026

YfiM is a lipid-linked oligosaccharide pyrophosphatase

Lipid-linked oligosaccharides (LLO), such as Lipid II and O-antigen precursors in bacteria, and dolichol-linked oligosaccharides (DLO) in eukaryotes, are universal metabolic intermediates required for cell wall assembly and protein glycosylation across all domains of life. The oligosaccharide moiety of LLO is synthesized through the sequential addition of various monosaccharides, a process catalyzed by a series of glycosyltransferases, to form the final mature structure. However, the existence of a homeostasis and quality control mechanism to degrade surplus and aberrant LLO has long remained a mystery. Although we recently identified the yeast LLP1 as an LLO pyrophosphatase involved in the homeostasis and quality control of DLO in fungi, the corresponding mechanism in bacteria and plants has remained elusive. Here, we identify YfiM and AT1G15900 as LLO pyrophosphatases in Escherichia coli and Arabidopsis thaliana, respectively. Phylogenetic profiling revealed that while YfiM is broadly conserved among Gram-negative bacilli, its plant ortholog displays a unique lineage-specific distribution shaped by selective gene loss. In vitro, purified YfiM and AT1G15900 exhibit LLO pyrophosphatase activity. In vivo, deletion mutant of yfiM caused vancomycin sensitivity in E. coli. Conversely, overexpression of YfiM triggered osmotic lysis under hypotonic conditions. Moreover, overexpression of YfiM in an outer membrane-compromised strain partially rescued detergent-chelator sensitivity, revealing a compensatory envelope stress response. Together, our findings uncover an ancient, conserved cross-kingdom system for LLO quality control bridging prokaryotes and plants.

microbiology↗

bioRxiv2026

Calibrated ADAMTS13 inhibition prevents cardiovascular shear coagulopathy

Mechanical circulatory support essential for managing severe heart failure frequently triggers bleeding complications, driven by shear stress-induced over-proteolysis of von Willebrand factor (VWF) by ADAMTS13. Inhibiting ADAMTS13 presents a rationale to treat this condition, known as acquired von Willebrand syndrome (AVWS). However, conventional therapeutic strategies remain limited due to the risk of triggering thrombotic thrombocytopenic purpura. Here we show that HA10, a humanized anti-ADAMTS13 antibody, preserves a residual level of ADAMTS13 activity above the thrombosis-associated threshold. Multimodal structural and biophysical analyses--including NMR, SAXS, and SANS--revealed that HA10 bound to the disintegrin-like domain of ADAMTS13, dynamically competing with VWF while leaving 10- 20% residual enzymatic activity. The therapeutic efficacy and safety of HA10 were verified in non-human primate models of AVWS. Our findings establish a novel paradigm of enzymatic calibration rather than complete blockade, offering a mechanistically targeted and safe therapeutic approach for cardiovascular bleeding.

biochemistry↗

bioRxiv2026

Whole-genome duplication underlies conserved sexually biased expression of meiotic cohesin genes unique to the teleost fish lineage

Meiosis is a fundamental process in producing both sperm and eggs, yet recombination landscapes often exhibit sexual differences, known as heterochiasmy. Since meiotic proteins are generally expressed in both sexes, the molecular mechanism driving heterochiasmy remains elusive. The -kleisin subunit gene of meiotic cohesin, Rec8, is expressed bisexually in mammals, while its putative teleost ortholog, rec8a, is expressed in a female-biased manner, presumably due to the presence of its paralog originating from the teleost-specific whole-genome duplication (TGD). Here, we elucidated the evolutionary history and expression dynamics of -kleisin genes across teleost lineages. Through comprehensive phylogenetic and synteny analyses, we revealed that major teleost lineages retain two copies of rec8 and rad21, with rec8 loci experiencing drastic chromosomal rearrangements immediately after the TGD. Using in situ hybridization and single-cell transcriptome data in medaka and zebrafish, we demonstrated a conserved sexually biased expression pattern: rec8a is predominantly female-biased, whereas rec8b exhibits male-biased expression during gametogenesis. Furthermore, comparative epigenetic analyses revealed that the conserved sexually biased expression is driven by lineage-specific cis-regulatory elements, rather than conserved ones. Motif analyses imply that regulatory rewiring by transcription factors, including foxl2l in particular, might have played a crucial role in the establishment and maintenance of this paralog divergence. Our findings highlight how whole-genome duplication and subsequent genomic and epigenetic rewiring subdivided the bisexual function of rec8, offering insights into sexually distinct meiotic regulation. HighlightsO_LITeleosts possess a unique -kleisin repertoire originating from the TGD. C_LIO_LITeleost rec8 paralogs exhibit conserved sex-biased expression during meiosis. C_LIO_LIDrastic genomic rearrangements after the duplication rewired the teleost rec8 loci. C_LIO_LIThe conserved expression pattern is governed by lineage-specific CREs. C_LIO_LIThose CREs harbor similar types of TFBSs such as Fox-family TFs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/731870v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c8c84dorg.highwire.dtl.DTLVardef@1d65668org.highwire.dtl.DTLVardef@c2d732org.highwire.dtl.DTLVardef@1be54a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Developmental Biology↗

bioRxiv2026

Assembly-pathway regulation dictates pH-responsive actuation in the R-body protein machinery

Refractile bodies (R-bodies) are protein assemblies that form tightly rolled morphologies in cells and undergo large-scale extension into long spirals in response to environmental stimuli. The type 51 R-body, the focus of this study, is assembled from four Reb proteins and undergoes a rapid repeatable [~]50-fold extension. Although R-bodies were first described more than 70 years ago, it has remained unclear how RebC and RebD contribute to the formation and function of this four-protein machinery even though RebA and RebB have been proposed as its major components. We characterized the wild-type R-body (Rb_WT) and a series of reb gene knockout mutants by combining in-cell, biochemical analyses, small-angle X-ray scattering (SAXS) and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. We found that RebD is incorporated into Rb_WT as a minor component, while RebC is not detectably incorporated into the final assembly. Mutants lacking either RebA or RebB still form roll-like assemblies but lack pH-dependent extension. In contrast, mutants lacking RebC and/or RebD exhibit a substantially lower propensity for roll formation and instead undergo off-pathway aggregation with increased {beta}-sheet content. SAXS analyses further indicate that roll-like morphology does not ensure formation of the ordered lamellar architecture characteristic of functional Rb_WT. Thus, pH-responsive R-body actuation is dictated not simply by the major proteins that constitute the final architecture, but by a regulated assembly pathway that builds the ordered lamellar architecture required for actuation. These findings establish assembly-pathway regulation as a key principle for constructing dynamic protein architectures capable of stimuli-responsive mechanical actuation.

biophysics↗

bioRxiv2026

Shark sexing from forensic, archival, and developmental samples using sex-linked DNA markers

The effective management of threatened shark populations relies on accurate demographic data, particularly operational sex ratios. While sex identification in intact shark bodies is straightforward through the presence of external male organs, namely claspers, it remains impossible for processed fins in the illegal wildlife trade, early-stage embryos in breeding programs, or archived tissue fragments and blood samples where morphological traits are lost. Here, we present a robust molecular sexing framework leveraging recently identified sequences from shark sex chromosomes, consistently organized in the XY system, to our current knowledge. Our approach consists of two distinct methodologies tailored to the the current identification status of sex chromosome sequences in the target species. For the whale shark Rhincodon typus and the brownbanded bamboo shark Chiloscyllium punctatum, we employed end-point PCR assays targeting male-specific Y-linked markers. For the cloudy catshark Scyliorhinus torazame, we developed a quantitative PCR (qPCR) assay targeting differential X chromosome dosage. In this dosage-based system, females (XX) are distinguished by an amplification profile approximately one cycle earlier than males (XY). By integrating X-linked dosage quantification, our framework provides a critical internal control that significantly enhances reliability, allowing researchers to distinguish true females from PCR failures. This toolkit offers a versatile solution for diverse applications, ranging from the study of sex determination mechanisms in pre-phenotypic embryos to the reconstruction of sex ratios from space-constrained tissue archives and global wildlife forensics, thereby contributing to the comprehensive conservation of shark biodiversity.

ecology↗

bioRxiv2025

Tracing genome size dynamics in sharks and rays with inclusive sequence analysis by the Squalomix Consortium

Genomes have maintained stable sets of protein-coding genes during evolution, while chromosome organization and genome size vary drastically. Changes in genome size are often attributed to variable amounts of repetitive sequences, including transposable elements. However, it remains poorly understood how such changes were accommodated while maintaining other genomic components. Elasmobranchs, including sharks, rays, and skates, exhibit high among-species variation of genome size and high within-species variation of chromosome length, offering a unique study system to address the question. In this study, we present the first whole genome sequences of the whitebelly skate with remarkably small genome size among elasmobranchs (2.2 Gb), and the red stingray. These chromosome-scale assemblies enabled the assessment of genomic compositions including centromeres and non-coding elements, which revealed notable profiles of tRNA loci and unbiased intragenomic distribution of transposons in elasmobranch genomes. Comparative analyses across these species revealed a shared genomic architecture characterized by correlations of intergenic and intronic sequence lengths with chromosome sizes, with repetitive element accumulation in elongated regions. We analyzed tandemly duplicated genes with high copy number variability. This genome-wide survey revealed the tendency for more frequent tandem gene duplications along with genome size expansion. We document the batoid HoxC cluster in the red stingray genome, which has undergone extensive repetitive element invasion and co-localizes with the HoxB cluster on a sex chromosome. Our study demonstrates an inclusive analysis encompassing both coding and non-coding regions, adaptable to more species in the taxon and a basis for molecular-level understanding on phenotypic diversity of elasmobranchs.

genomics↗

bioRxiv2025

Sharks and rays have the oldest vertebrate sex chromosome with unique sex determination mechanisms

Sex determination has been investigated across vertebrate lineages to reveal stepwise evolution of sex chromosomes and diversity of responsible molecular mechanisms. However, these studies hardly encompass cartilaginous fishes deeply isolated from the rest of vertebrates, which hinders the comprehensive view of vertebrate sex determination. Here, we produced chromosome-scale genome assemblies of egg-laying shark species and comparatively investigated genome sequences and transcriptome profiles across diverse cartilaginous fishes. Sex chromosome identification, supported by cytogenetic experiments, elucidated the homology of X chromosomes between sharks and rays as well as an extensively degenerating Y chromosome harboring no male-specific genes. These sex chromosomes scarcely included orthologs of previously documented sex determining genes. Transcriptomic analyses combined with histology of embryonic gonads revealed female-biased expressions of X-linked genes including those implicated in TGF-{beta} and IGF signaling pathways, which are attributed to their incomplete dosage compensation. Our findings indicate that sharks and rays share the oldest vertebrate sex chromosomes that originated around 300 million years ago and the dosage-dependent sex determination mechanism comprised of distinct molecules from other vertebrates. This study highlights the antiquity of sex chromosomes and uniqueness of sex determination mechanisms in sharks and rays, which advance our understanding on evolutionary plasticity of vertebrate sex determination.

evolutionary biology↗

bioRxiv2025

Arabidopsis TITAN LIKE is required for U12-type intron splicing, especially for AT-AC subtypes

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes more than 99% of all introns, whereas the U12-dependent spliceosome acts on only about 0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human CENATAC is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN LIKE (TTL), but its function in splicing remains unknown. Here, we generated TTL mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild-type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than that in a DROL1 mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

plant biology↗

bioRxiv2025

Temperature vaulting: A method for screening selective, slow- and tight-binding inhibitors targeting kinases in their non-native state

A polypeptide folds into its protein tertiary structure in the native state through a folding intermediate in the non-native state. The transition between these states is thermodynamically driven. A folding intermediate of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) autophosphorylates intramolecularly, whereas DYRK1A in the native state no longer catalyzes this reaction. The alteration in substrate specificity suggests a conformational transition of DYRK1A during its folding process. Consistent with this hypothesis, we identified FINDY (1), which inhibits the intramolecular autophosphorylation but not the intermolecular phosphorylation, suggesting that the folding intermediate possesses an alternative inhibitor-binding site. Meanwhile, it remains an issue that the methods for approaching the alternative binding site require an intricate assay tailored to the individual target. Here we show a method, designated as "temperature vaulting," for screening the non-native-state-targeted inhibitors of DYRK1A. Transient heating of recombinant DYRK1A protein drove the reversible transition between the native state and the non-native state targeted by FINDY (1). At physiological temperature, FINDY (1) slowly bound to the DYRK1A protein. These results indicate that transient heating accelerates the slow-binding process by assisting the protein to overcome the high-energy barrier leading to the target non-native state. The energy barrier also slowed down the dissociation process, resulting in tight binding between DYRK1A and FINDY (1). Furthermore, this study suggests that the dissociation rate underlies the inhibition selectivity of FINDY (1) between DYRK1A and its family kinase DYRK1B. This method enables the identification of slow- and tight-binding inhibitors that have been missed in conventional assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/632643v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@be5661org.highwire.dtl.DTLVardef@1925beorg.highwire.dtl.DTLVardef@fceb5borg.highwire.dtl.DTLVardef@bfbf91_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSA temperature vaulting method facilitated the screening of a slow- and tight-binding inhibitor targeting DYRK1A in the non-native state. A structure-activity relationship study of FINDY (1) demonstrated that a slight alteration in chemical structure impacts the inhibition kinetics. A molecular dynamics simulation of DYRK1A revealed binding-ready conformations for FINDY (1). The dissociation rate may underlie the inhibition selectivity of FINDY (1) between DYRK1A and DYRK1B in cellular systems.

biophysics↗

bioRxiv2024

Conserved loop of a phase modifier endows protein condensates with fluidity

Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion in C9orf72 trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherin{beta}2 (Kap{beta}2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kap{beta}2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kap{beta}2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kap{beta}2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight into C9orf72-ALS related to age and repeat expansion.

biochemistry↗

bioRxiv2024

A newly-identified mini-hairpin shaped nascent peptide blocks translation termination by a novel mechanism.

Protein synthesis by ribosomes not only produces functional proteins but also serves diverse functions depending on the coding amino acid sequences. Certain nascent peptides interact with the ribosome exit tunnel to arrest translation and modulate the expression of downstream genes or themselves. However, a comprehensive understanding of the mechanisms of such ribosome stalling and its regulation remains elusive. In this study, we systematically screened for unidentified ribosome arrest peptides through phenotypic evaluation, proteomics, and MS analyses, leading to the discovery of novel arrest peptides PepNL and NanCL in E. coli. Our cryo-EM study on PepNL revealed a unique arrest mechanism, in which the N-terminus of PepNL folds back towards the tunnel entrance to prevent the catalytic GGQ motif of release factor from accessing the peptidyl transferase center, causing translation arrest at the UGA stop codon. Furthermore, unlike other sensory arrest peptides that require an arrest inducer, PepNL uses tryptophan as an arrest releaser, where Trp-tRNA reads through the stop codon. Our findings illuminate the mechanism and regulatory framework of nascent peptide-induced translation arrest, paving the way for exploring regulatory nascent peptides.

molecular biology↗

bioRxiv2024

Pronounced expression of extracellular matrix proteoglycans regulated by Wnt pathway underlies the parallel evolution of lip hypertrophy in East African cichlids

Cichlid fishes inhabiting the East African Great Lakes, Victoria, Malawi, and Tanganyika, are textbook examples of parallel evolution, as they have acquired similar traits independently in each of the three lakes during the process of adaptive radiation. In particular, "hypertrophied lip" has been highlighted as a prominent example of parallel evolution. However, the underlying molecular mechanisms remain poorly understood. In this study, we conducted an integrated comparative analysis between the hypertrophied and normal lips of cichlids across three lakes based on histology, proteomics, and transcriptomics. Histological and proteomic analyses revealed that the hypertrophied lips were characterized by enlargement of the proteoglycan-rich layer, in which versican and periostin proteins were abundant. Transcriptome analysis revealed that the expression of extracellular matrix-related genes, including collagens, glycoproteins and proteoglycans, was higher in hypertrophied lips, regardless of their phylogenetic relationships. In addition, the genes in Wnt signaling pathway, which is involved in promoting proteoglycan expression, was highly expressed in both the juvenile and adult stages of hypertrophied lips. Our comprehensive analyses showed that hypertrophied lips of the three different phylogenetic origins can be explained by similar proteomic and transcriptomic profiles, which may provide important clues into the molecular mechanisms underlying phenotypic parallelisms in East African cichlids.

evolutionary biology↗

bioRxiv2023

Latent inter-organ mechanism of idiopathic pulmonary fibrosis unveiled by a generative computational approach

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people worldwide. While the molecular and cellular details of the IPF mechanism is emerging, our current understanding is centered around the lung itself. On the other hand, many human diseases are the products of complex multi-organ interactions. Hence, we postulate that a dysfunctional crosstalk of the lung with other organs plays a causative role in the onset, progression and/or complications of IPF. In this study, we employed a generative computational approach to identify such inter-organ mechanism of IPF. The approach works as follows: 1) To find unexpected relatedness of IPF to other diseases of non-lung organs and to identify molecular features that define such relatedness, 2) To identify differentially expressed genes between the lung tissues of IPF vs. those of non-IPF pulmonary disease patients, 3) To detect ligand-receptor relationships across multiple organs and their upstream and downstream signaling pathways in 1) and 2), 4) To generate a map of the inter-organ IPF mechanism with the molecular and cellular resolution. This approach found unexpected molecular relatedness of IPF to neoplasm, diabetes, Alzheimers disease, obesity, atherosclerosis, and arteriosclerosis. Furthermore, as a potential mechanism underlying this relatedness, we uncovered a putative molecular crosstalk system across the lung and the liver. In this inter-organ system, a secreted protein, kininogen 1, from hepatocytes in the liver interacts with its receptor, bradykinin receptor B1 in the lung. This ligand-receptor interaction across the liver and the lung leads to the activation of calmodulin pathways in the lung, leading to the activation of interleukin 6 and phosphoenolpyruvate carboxykinase 1 pathway across these organs. Furthermore, we retrospectively identified several pre-clinical and clinical evidence supporting this inter-organ mechanism of IPF. In conclusion, such feedforward and feedback system across the lung and the liver provides a unique opportunity for the development of the treatment and/or diagnosis of IPF. Furthermore, the result illustrates a generative computational framework for machine-mediated synthesis of mechanisms that facilitates and complements the traditional experimental approaches in biomedical sciences.

pathology↗

bioRxiv2022

Novel trans-translation-associated gene regulation revealed by prophage excision-triggered switching of ribosome rescue pathway

Escherichia coli has multiple pathways to release a nonproductive ribosome complex stalled at the 3 end of nonstop mRNAs: SsrA RNA-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and thus its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the situations in which ArfA is expressed to rescue the nonproductive complexes have not been elucidated. Here, we demonstrated that the excision of the CP4-57 prophage adjacent to the E. coli ssrA gene leads to the inactivation of SsrA RNA and switches the primary rescue pathway from trans-translation to the ArfA/RF2 pathway. A comparative quantitative proteomic analysis revealed that the switching of the rescue pathway rearranges not only the proteome landscape in E. coli cells but also the phenotype, such as motility. Among the proteins with significantly increased abundance in the ssrA-inactivated cells, we found ZntR, whose mRNA is transcribed together as the upstream part of the nonstop arfA mRNA. Further analysis revealed that the translation of the nonstop ORF of arfA triggered the repression of the upstream zntR ORF, via trans-translation-coupled exonucleolytic (RUTEX) degradation by a polynucleotide phosphorylase. These results provide a novel example of trans-translation-dependent regulation, and shed new light on the physiological roles of prophages in gene expression.

genetics↗

bioRxiv2022

A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains; i.e., polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified [~]800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods for profiling nascent chains such as ribosome profiling. Significance StatementIn the central dogma of biology, RNA and protein are usually regarded as two completely independent molecular species. However, they are combined into a single species called peptidyl-tRNA (pep-tRNA) during the translation process in the ribosome. Despite the importance of pep-tRNAs as precursors of all proteins in the cell, a general method to analyze pep-tRNAs on a large scale was lacking. Taking advantage of the properties of pep-tRNAs as RNA and protein, we developed a method to enrich the pep-tRNAs by organic solvent extraction and silica column separation. The method, termed PETEOS, not only provides a unique approach to examine the nascent state of proteins but also may be effective in capturing snapshots of translation status in the cell.

biochemistry↗

bioRxiv2022

Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes

Robust translation elongation of any given amino acid sequence is a prerequisite to shape proteomes. Nevertheless, nascent peptides could destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains stochastically can induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in the N-terminal regions could prematurely terminate translation, producing premature products as peptidyl-tRNA species. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we found many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk. Indeed, a bioinformatics analysis revealed that the N-terminal regions of ORFs avoid D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

biochemistry↗

bioRxiv2021

Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

immunology↗

bioRxiv2021

Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

biochemistry↗