Biology subjects

Mito, M.

Publications and source records attributed to Mito, M..

At least 19 recordsLinked to original sources

bioRxiv2026

RNA-dependent RNA amplification as a strategy to perform tRNA sequencing on low-input samples

Transfer RNAs (tRNAs) are key molecules that deliver amino acids to the translating ribosome according to their cognate codon encoded in messenger RNA (mRNA). Due to the modified nature of tRNA nucleotides, accurate tRNA quantification can be tedious, especially when dealing with small sample inputs. Here, we took advantage of an RNA-dependent RNA amplification method using T7 polymerase to quantify tRNA abundance in low biological input. Our method, called T7 High-resolution original RNA (Thor)-tRNA-Seq, showed reproducible and quantitative measurement of low tRNA inputs. Thus, our Thor-tRNA-Seq is a robust and reliable approach for the quantification of tRNA in samples with small and precious biological material.

molecular biology↗

bioRxiv2025

Revisiting the in vivo Functions of Coilin in Mice

Cajal bodies (CBs), first identified in the early 20th century, are classic nuclear structures organized primarily by the protein coilin. Although CBs have long been implicated in snRNA modification and maturation, their precise roles in mice remain unclear. Here, we revisit the functions of CBs by using genome editing to generate mice that lack nearly the entire coilin protein, retaining only the first seven amino acids. Mass-spectrometry analyses showed reduced 2'-O-methylation of U2 snRNA in coilin mutant liver; however, these reductions had minimal impact on overall gene expression and pre-mRNA splicing patterns. Additionally, detailed analysis recapitulated previously reported testicular size reductions and newly revealed decreased proportions of high-quality sperm. We also identified novel coilin-containing condensates distinct from genuine CBs in the nuclear pocket of elongating spermatids. Our findings implicate that coilin orchestrates membraneless organelles in various compartments, playing multifunctional roles beyond classical CB formation.

molecular biology↗

bioRxiv2025

Ultra-parallel ribosome profiling platform 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 and a large number of samples have presented analytic challenges. Here, we developed the semi-automated platform of Ribo-Seq and Disome-Seq, which allowed us to assess the translation status from a vast collection of samples with reduced amounts in a plate format. Through an siRNA-mediated knockdown screen for ribosome-associated proteins, this technique identified factors that (i) mediate regulation via RNA elements such as the TOP motif, (ii) assist efficient ribosome recycling, (iii) support ribosome-associated quality control (RQC), (iv) enhance translation elongation across inhibitory G-quadruplex sequences, and (v) repress mitochondrial translation. The application to human-derived samples revealed that stop codon readthrough and mitochondrial translation deficiency are associated with severe symptoms in COVID-19. Our approach provides a versatile option to investigate the translatome in a highly parallel manner.

molecular biology↗

bioRxiv2025

eIF4G2-Mediated Translation Initiation of Histone Modifiers Is Essential for Intestinal Stem Cell Maintenance and Differentiation

eIF4G2 (also known as NAT1, p97, or DAP5) is an evolutionally conserved protein homologous to the C-terminal two-thirds portion of eukaryotic translation initiation factor (eIF) 4G. Despite its abundant and ubiquitous expression, the physiological and pathological functions of eIF4G2 are poorly understood. Here, we show that acute loss of eIF4G2 in adult mice results in rapid weight loss with abnormalities in multiple organs, including impaired maintenance and differentiation of intestinal stem cells. Genome-wide ribosome profiling revealed that eIF4G2 is critical for the translation of key histone modification proteins involved in intestinal stemness. Our study underscores the importance of eIF4G2-mediated translation initiation in multicellular organisms.

cell biology↗

bioRxiv2025

Mitochondrial translation termination, recycling, reinitiation, and rescue for in-frame and out-of-frame contexts

Because mitochondria diverged from a bacterial ancestor during evolution, the mitochondrial protein synthesis system includes both mRNAs and translation factors with unique characteristics. However, the molecular mechanisms underlying translation termination, recycling, and quality control remain unclear. Here, via high-resolution mitochondrial Ribo-Seq and Disome-Seq, we revealed: the specificity of release factors for different kinds of stop codons; the role of mtRF1 in vertebrates, which do not have noncanonical stop codons in their main ORFs; the recycling-coupled translation of internal ORFs; and the rescue of mitoribosomes in the early elongation stage. mtRF1L is a universal release factor that recognizes all stop codons, whereas mtRF1 recognizes only AGA/AGG noncanonical stop codons. Additionally, mtRF1 terminates the translation of out-of-frame ORFs that end with AGA/AGG. We also found that mtRRF and mtIF3 are required for mitoribosome recycling on stop codons and for the reinitiation of internal ORF translation. Mitoribosomes that stall at start codons are major substrates of the rescue factors ICT1, mtRF-R, and mtRES1. Moreover, HEMK1-mediated methylation of release factors enhances the termination reaction on stop codons. Our results provide insights into the mitoribosome dynamics that are associated with the completion of protein synthesis.

molecular biology↗

bioRxiv2024

Giant virus creates subcellular environment to overcome codon-tRNA mismatch

Codon usage consonant with the cellular tRNA pool is important for efficient translation. However, many eukaryotic viruses, including amoeba-infecting mimiviruses, have codon usage that is largely deviated from that of their host, despite using the host machinery for translation. This raises the question of how these viruses cope with the mismatch between tRNA supply and demand. Here we show that Acanthamoeba castellanii mimivirus generates a subcellular area in the host cells to translate virus mRNAs. A combination of genome-wide ribosome profiling and RNA sequencing showed that ribosomes traversed along viral mRNAs with fewer pausing events than were observed on amoeba mRNAs. Frequently used codons in viral mRNAs had higher tRNA accessibility than the same type of codons in amoeba mRNAs. tRNA sequencing showed that the tRNA pool was not greatly altered during the infection even though the virus encodes tRNA genes. Instead, by in situ labelling, we found that viral mRNAs and newly synthesized proteins were localized at the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation assists the virus in overcoming the mismatch between tRNA supply and demand.

microbiology↗

bioRxiv2024

Sense codon-misassociated eRF1 elicits widespread ribosome stalling and induction of quality control

The rescue pathway of stalled ribosomes and ribosome-associated quality control (RQC) serves as a surveillance system for aberrant translation that senses ribosome collisions. Although the molecular mechanism has been extensively studied, the endogenous contexts of ribosome collision rescued by the system are poorly understood. Here, beginning with a study of the codon specificity of the eukaryotic translation termination factor eRF1, we show that transient binding of eRF1 to the UUA sense codon leads to ribosome collision and provides a constitutive source of rescue substrates in humans. eRF1-selective Monosome-Seq and Disome-Seq revealed that eRF1 was recruited not only to stop codons but also to near-cognate sense codons, including the UUA codon. The eRF1 on UUA codons delays translation elongation but does not trigger the termination reaction. Remarkably, Disome-Seq with the depletion of ASCC3 and 4EHP, key factors for the ribosome rescue and translation initiation repression, showed that ribosomes stalled at UUA codons consist of a subpopulation suppressed by those factors. Failure of ribosome collision repression by ASCC3-4EHP triggers stress response, such as expression of the stress-induced transcription factor ATF3. This study highlights the impact of sense codon misrecognition by the termination factor on translation homeostasis in human cells.

molecular biology↗

bioRxiv2024

Selective Translation Orchestrates Key Signaling Pathways in Primed Pluripotency

Pluripotent stem cell (PSC) identities, such as differentiation and infinite proliferation, have long been understood within the frameworks of transcription factor networks, epigenomes, and signal transduction, yet remain unclear and fragmented. Directing attention toward translational regulation, as a bridge between these events, promises to yield new insights into previously unexplained mechanisms. Our functional CRISPR interference screening-based approach revealed that EIF3D maintains primed pluripotency through selective translational regulation. The loss of EIF3D disrupts the balance of pluripotency-associated signaling pathways, impairing primed pluripotency. Moreover, we discovered that EIF3D ensures robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, selective translation by EIF3D tunes the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. Therefore, this study establishes a paradigm for selective translational regulation as a defining feature of primed PSC identity.

cell 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

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

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

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↗

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

eIF2B-capturing viral protein NSs suppresses the integrated stress response

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(P)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotides on unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. Our cryo-electron microscopy (cryo-EM) analysis revealed that SFSV NSs binds to the -subunit of eIF2B in a competitive manner with eIF2(P). Together with SFSV NSs, eIF2B exhibits normal nucleotide exchange activity even in the presence of eIF2(P). A genome-wide ribosome profiling analysis clarified that SFSV NSs in human cultured cells attenuates the ISR. Furthermore, SFSV NSs exhibited neuroprotective effects against the ISR-inducing stress. Since the ISR inhibition is beneficial in various neurological disease models, SFSV NSs is promising as a therapeutic ISR inhibitor.

molecular biology↗

bioRxiv2020

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Chemical splicing modulators that bind to the spliceosome have provided an attractive venue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. Yet, the biological effect of proteins containing translated intron sequences remains unclear. Here we identified a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) using genome-wide ribosome profiling and bio-orthogonal non-canonical amino-acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through JNK phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

molecular biology↗