Biology subjects

Iwasaki, S.

Publications and source records attributed to Iwasaki, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Translation variation across genetic backgrounds reveals a post-transcriptional buffering signature in yeast

Gene expression is known to vary among individuals, and this variability can impact the phenotypic diversity observed in natural populations. While the transcriptome and proteome have been extensively studied, little is known about the translation process itself. Here, we therefore performed ribosome and transcriptomic profiling on a genetically and ecologically diverse set of natural isolates of the Saccharomyces cerevisiae yeast. Interestingly, we found that the Euclidean distances between each profile and the expression fold changes in each pairwise isolate comparison were higher at the transcriptomic level. This observation clearly indicates that the transcriptional variation observed in the different isolates is buffered through a phenomenon known as post-transcriptional buffering at the translation level. Furthermore, this phenomenon seemed to have a specific signature by preferentially affecting essential genes as well as genes involved in complex-forming proteins, and low transcribed genes. We also explored the translation of the S. cerevisiae pangenome and found that the accessory genes related to introgression events displayed similar transcription and translation levels as the core genome. By contrast, genes acquired through horizontal gene transfer events tended to be less efficiently translated. Together, our results highlight both the extent and signature of the post-transcriptional buffering.

systems biology↗

bioRxiv2023

Pateamine A mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only limited examples have been reported to date. Here, we show that pateamine A (PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, on GNG motifs. Through a systematic comparison of multiple eIF4A inhibitors by ribosome profiling, we found that PatA has unique mRNA selectivity in translation repression. Unbiased Bind-n-Seq revealed that PatA-targeted eIF4A exhibits a sequence preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. In silico simulation, combination of classical molecular dynamics simulation and quantum chemical calculation, and the subsequent development of an inactive PatA derivative revealed that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified DDX3, another DEAD-box protein, as an alternative target of PatA, showing the same effect as on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and mRNA-selective inhibition of protein synthesis by small-molecule compounds.

molecular biology↗

bioRxiv2023

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) lost the elongated spermatids and mature sperm, which was fully rescued by a Mettl1-transgene expression, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO resulted in a loss of m7G modification on a subset of tRNAs and a decreased level of tRNA expression. Strikingly, overexpression of the translational elongation factor, EF11, which can compete with the rapid tRNA decay (RTD) pathway in S. cerevisiae, significantly counteracted the sterility of Mettl1-KO males, supporting a critical role of m7G modification of tRNAs in spermatogenesis. Ribosome profiling showed that Mettl1-KO led to the ribosome stalling at codons decoded by tRNAs that were reduced in expression. Mettl1-KO also significantly reduced the translation efficiency of genes involved in elongated spermatid formation and sperm stability. These findings reveal a developmental role for m7G tRNA modifications and indicate that m7G modification-dependent tRNA stability differs among tissues.

molecular biology↗

bioRxiv2023

Complexity and dynamics of in organello translation landscape assessed by high-resolution mitochondrial ribosome profiling

Since mitochondrial translation serves the essential subunits of the OXPHOS complex that produces ATP, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and revealed the intricate regulation of mammals in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at the anticodon stem loop in mt-tRNAs, including all possible modifications at the 34th position, by deleting the corresponding enzymes and harnessing patient-derived cells. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.

molecular biology↗

bioRxiv2023

No-go decay dampens zinc finger protein mRNAs in vertebrates

Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 downregulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. We further showed that mRNAs encoding C2H2-ZF were degraded in a ZNF598-dependent manner in HEK293T cells. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.

molecular biology↗

bioRxiv2023

Absolute calibration of ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to determine overall ribosome numbers in transcripts. Here, we overcame these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches measures the absolute number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method revealed the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy transforms ribosome profiling technique from relative to absolute quantification of translation.

molecular biology↗

bioRxiv2023

Translational regulation enhances distinction of cell types in the nervous system

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically-defined cells in the Drosophila brain, we here revealed substantial posttranscriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias towards the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open reading frames (uORFs) in the 5 leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

neuroscience↗

bioRxiv2023

CRISPRδ: dCas13-mediated translational repression for accurate gene silencing in mammalian cells

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)Cas13 systems, have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPRCas13). Thus, a more specific method of gene knockdown is needed. Here, we developed "CRISPR{delta}", an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent or internal ribosome entry site (IRES)-dependent translation. Strikingly, genome-wide ribosome profiling revealed the extremely high gene knockdown specificity of CRISPR{delta}. Moreover, fusion of a translational repressor to dCas13 ensured further improvement of the knockdown efficacy. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

molecular biology↗

bioRxiv2023

Gravitational and mechanical forces drive mitochondrial translation through the cell adhesion-FAK axis

Life on Earth has evolved in a form suitable for the gravitational force of 1 x g. Although the pivotal role of gravity in gene expression has been revealed by multiomics approaches in space-flown samples and astronauts, the molecular details of how mammalian cells harness gravity have remained unclear. Here, we showed that mitochondria utilize gravity to activate protein synthesis within the organelle. Genome-wide ribosome profiling revealed reduced mitochondrial translation in mammalian cells and Caenorhabditis elegans under both microgravity at the International Space Station and simulated microgravity in a 3D-clinostat on the ground. We found that attenuation of cell adhesion through laminin-integrin interactions causes the phenotype. The downstream signaling pathway including FAK, RAC1, PAK1, BAD, and Bcl-2 family proteins in the cytosol, and mitochondrial fatty acid synthesis (mtFAS) pathway in the matrix maintain mitochondrial translation at high level. Mechanistically, a decreased level of mitochondrial malonyl-CoA, which is consumed by activated mtFAS, leads to a reduction in the malonylation of the translational machinery and an increase in the initiation and elongation of in organello translation. Consistent with the role of integrin as a mechanosensor, we observed a decrease in mitochondrial translation via the minimization of mechanical stress in mouse skeletal muscle. Our work provides mechanistic insights into how cells convert gravitational and mechanical forces into translation in an energy-producing organelle.

molecular biology↗

bioRxiv2023

Thor-Ribo-Seq: ribosome profiling tailored for low input with RNA-dependent RNA amplification

Translation regulation plays a pivotal role in the diversification of gene expression and the response to intra- and extracellular environmental cues. Ribosome profiling (or Ribo-Seq) serves as a sensitive, quantitative, comprehensive, and data-rich technique to survey ribosome traversal across the cellular transcriptome. However, due to the intricacy of library preparation, applications to low input have presented analytic challenges. To overcome this issue, here we developed Thor-Ribo-Seq, a ribosome profiling method tailored for low input. Thor-Ribo-Seq harnesses RNA-templated RNA transcription to linearly amplify ribosome footprints, assessing ribosome traversal at codon resolution with limited artifacts. This highly sensitized ribosome profiling approach provides a versatile option to investigate the translatome in precious samples.

molecular biology↗

bioRxiv2022

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier of antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocalgates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to Ophiocordyceps genus and its eIF4A, a molecular target of rocaglates, contains a amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered Colletotrichum orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

molecular biology↗

bioRxiv2022

Species-Specific Formation of Paraspeckles in Intestinal Epithelium Revealed by Characterization of NEAT1 in Naked Mole-rat

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2. The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2, that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

molecular biology↗

bioRxiv2022

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

molecular biology↗

bioRxiv2021

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

microbiology↗

bioRxiv2021

Detection of the 40-Hz Auditory Steady-state Response with Optically Pumped Magnetometers

Magnetoencephalography (MEG) is a functional neuroimaging technique that noninvasively detects the brain magnetic field from neuronal activations. Conventional MEG measures brain signals using superconducting quantum interference devices (SQUIDs). SQUID-MEG requires a cryogenic environment involving a bulky non-magnetic dewar and the consumption of liquid helium, which restricts the variability of the sensor array and the gap between the cortical sources and sensors. Recently, miniature optically pumped magnetometers (OPMs) have been developed and commercialized. OPMs do not require cryogenic cooling and can be placed within millimeters from the scalp. In the present study, we arranged six OPM sensors on the temporal area to detect auditory-related brain responses in a two-layer magnetically shielded room. We presented the auditory stimuli of 1-kHz pure-tone bursts with 200-ms duration and obtained the M50 and M100 components of auditory evoked fields. We delivered the periodic stimuli with a 40-Hz repetition rate and observed the gamma-band power changes and inter-trial phase coherence of auditory steady-state responses at 40 Hz. We found that the OPM sensors have a performance comparable to that of conventional SQUID-MEG sensors, and our results suggest the feasibility of using OPM sensors for functional neuroimaging and brain-computer interface applications.

neuroscience↗

bioRxiv2021

A specific eIF4A paralog facilitates LARP1-mediated translation repression during mTORC1 inhibition

Eukaryotic translation initiation factor (eIF) 4A -- a DEAD-box RNA-binding protein -- plays an essential role in translation initiation. Two mammalian eIF4A paralogs, eIF4A1 and eIF4A2, have been assumed to be redundant because of their high homology, and the difference in their functions has been poorly understood. Here, we show that eIF4A1, but not eIF4A2, enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA-immunoprecipitation sequencing (RIP-Seq) of the two eIF4A paralogs revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTOR inhibition. This biased interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that the deletion of EIF4A1, but not EIF4A2, rendered the translation of TOP mRNAs resistant to mTOR inactivation. Moreover, eIF4A1 enhances the affinity between TOP mRNAs and LARP1 and thus ensures stronger translation repression upon mTORC1 inhibition. Our data show that the distinct protein interactions of these highly homologous translation factor paralogs shape protein synthesis during mTORC1 inhibition and provide a unique example of the repressive role of a universal translation activator.

molecular biology↗

bioRxiv2021

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

genetics↗

bioRxiv2021

METTL18-mediated histidine methylation on RPL3 modulates translation elongation for proteostasis maintenance

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a substrate for the modification. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traverse on tyrosine codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the {tau}-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation tuning. Indeed, genome-wide ribosome profiling revealed suppressed ribosomal translocation at tyrosine codons by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a "ribosome code" that ensures proteome integrity in cells.

molecular biology↗