Biology subjects

Niizuma, K.

Publications and source records attributed to Niizuma, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Long read multi-omics sequencing reveals DNA-to-RNA evolutionary remodeling trajectories of recurrent astrocytoma

Astrocytoma recurrence is shaped by genetic, epigenetic and transcriptional evolution, but how DNA remodeling is propagated into full-length RNA isoforms and coding consequences remains poorly resolved. Here we applied paired PacBio HiFi whole-genome sequencing and Kinnex full-length RNA sequencing to matched primary and recurrent astrocytoma from six patients, with matched blood controls. Recurrent tumors preserved core glioma driver identity while acquiring patient-specific remodeling across somatic variants, copy number, structural variation, loss of heterozygosity, haplotype imbalance and DNA methylation. Long-read transcriptomics revealed extensive recurrence-associated gene-expression, isoform-usage, differential transcript-usage and predicted ORF/protein-fate remodeling beyond gene-level expression. A denominator-aware integration framework showed that copy-number changes provide broad RNA-dosage links, whereas expressed somatic variants, allele/haplotype-specific transcript usage, methylation-linked isoform remodeling and structural variants generate more focused DNA-RNA-ORF chains. These findings establish longitudinal long-read multi-omics as a framework for prioritizing patient-specific remodeling trajectories and identifying targets for precision medicine in recurrent astrocytoma.

cancer biology↗

bioRxiv2025

Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

Diffuse midline glioma (DMG) is a near-universally lethal form of pediatric high-grade glioma, driven by neuronal activity-regulated paracrine signaling and synaptic integration of malignant cells into neural circuits. In turn, DMG increases neuronal excitability, augmenting neuron-to-glioma signaling. In the healthy brain, microglia, the resident immune cells of the central nervous system (CNS), regulate neuronal excitability and synaptic connectivity. However, the role of microglia in promoting tumor-associated hyperexcitable neural networks in glioma remains unknown. Here, we investigate the activity-regulated engulfment of neuronal synapses by microglia in both healthy and glioma-bearing mice, and further explore how glioma cells alter microglia-mediated circuit refinement, contributing to pathogenic neuronal hyperexcitability. Microglia-mediated circuit refinement in the glioma microenvironment was characterized through synaptic engulfment analysis of both excitatory and inhibitory synapses by microglia in healthy mice and patient-derived DMG xenograft models, paired with optogenetic stimulation in the neocortex. We found that glutamatergic neuronal activity in the healthy brain increased excitatory synaptic engulfment by microglia in a previously unappreciated negative feedback mechanism that may guard against hyperexcitability. In contrast, this activity-regulated increase in excitatory synaptic engulfment was abrogated in DMG-infiltrated brains. Instead, inhibitory synaptic engulfment was significantly increased in DMG in response to glutamatergic neuronal activity. Together, these dysregulated synaptic engulfment mechanisms may create imbalance in the excitatory to inhibitory (E:I) synapse ratio predicted to increase neuronal excitability. Complementary single-nuclei sequencing studies revealed concordant tumor-specific, activity-regulated changes in microglia-neuron signaling showing reduced expression of excitatory synaptic refinement gene programs in microglia, potentially mediating the aberrant synaptic engulfment observed in DMG. These findings reveal novel cancer-neuron-immune interactions in DMG and provide an opportunity to potentially modulate tumor-associated neuronal hyperexcitability by targeting aberrant microglial synaptic engulfment. HIGHLIGHTSO_LIExcitatory synaptic engulfment by microglia increases in a glutamatergic neuronal activity-dependent manner in the healthy brain. C_LIO_LIIn the glioma-infiltrated brain, this feedback regulatory mechanism is inverted, with loss of activity-regulated excitatory synaptic engulfment and aberrant increased inhibitory synaptic engulfment. C_LIO_LITranscriptomic changes in glioma-associated microglia contribute to differential excitatory and inhibitory synaptic engulfment, with reduced excitatory synaptic refinement gene programs in tumor-associated microglia. C_LIO_LIActivity-regulated soluble factors drive this imbalanced excitatory-inhibitory postsynaptic engulfment by glioma-associated microglia. C_LI

neuroscience↗

bioRxiv2025

Multiscale modeling of blood circulation with cerebral autoregulation and network pathway analysis for hemodynamic redistribution in the vascular network with anatomical variations and stenosis conditions

Cerebral hemodynamics is fundamentally regulated through the Circle of Willis (CoW), which redistribute flow via communicating arteries to stabilize perfusion under anatomical variations and vascular stenosis. In this study, a multiscale circulation model was developed by coupling a multiscale systemic hemodynamic framework with cerebral arterial network reconstructed from medical imaging. The model integrates a cerebral autoregulation mechanism (CAM) and enables quantitative simulation of flow redistribution across the CoW under normal, anatomically varied, and pathologically narrowing (stenosis) conditions. Baseline simulations at normal states reproduced physiological flow distributions in which the communicating arteries remained nearly inactive, showing negligible across-flow and agreement with clinical measurements, while two anatomical variations revealed distinct collateral activation patterns: the anterior communicating artery (ACoA) acted as the earliest and most sensitive functional collateral pathway, whereas the posterior communicating arteries (PCoAs) exhibited structure-dependent engagement. Progressive stenosis modelling further demonstrated the transition from a complete CoW to a fetal-type posterior cerebral artery (PCA) configuration, with early ACoA flow reversal followed by the ipsilateral PCoA activation, in agreement with experimental and transcranial Doppler observations. We further present a path-based quantitative analysis of source-to-sink flow contributions to quantitatively illustrates how the cerebral vascular network dynamically reconfigures collateral pathways in response to structural changes. Overall, the proposed framework provides a physiologically interpretable and image-informed tool for investigating cerebral flow regulations through the functional collaterals within the CoW, offering potential for clinical applications in the diagnosis, prognosis, and treatment planning of cerebrovascular diseases.

biophysics↗

bioRxiv2025

ALKBH1 drives codon-biased, pro-oncogenic translation and tumor microenvironment remodeling in glioma via tRNA wobble oxidation

Gliomas rely on translational plasticity to sustain heterogeneity, stemness, and immune evasion. Here we identify the tRNA dioxygenase ALKBH1 as a central regulator of codon-biased translation and tumor microenvironment remodeling. LC-MS/MS profiling of patient tumors revealed enrichment of ALKBH1-mediated wobble cytidine oxidation modifications in high-grade gliomas. Genetic perturbation demonstrated that ALKBH1 overexpression slows proliferation in vitro but worsens survival in vivo by promoting glioma stem-like and neuronal states and suppressing anti-tumor immunity. Ribosome profiling showed that ALKBH1 establishes an A/T-ending codon-biased translational program, enhancing decoding of rare leucine codons (TTA/TTG) and driving synthesis of pro-stemness transcripts. Single-cell RNA-seq analyses further revealed that ALKBH1 promotes glioma heterogeneity and induces neuronal cell clusters and rewires intercellular communication ECM-related signaling while dampening immune pathways and reducing immune infiltration. These findings establish a mechanistic link between tRNA oxidation, codon bias, and glioma aggressiveness, positioning ALKBH1 as a potential therapeutic target for glioma.

cancer biology↗

bioRxiv2025

Third-nucleotide codon bias and synonymous codon bias define functional translational programs that shape human tissue and cancer proteomes.

BackgroundCodon usage bias is a universal feature of the genetic code, yet how synonymous codon bias or third-nucleotide codon bias (A/T-vs G/C-ending) shape translation and proteome composition across tissues and cancer remain unclear. ResultsUsing comparative genomics between human and rodent coding sequences, we uncovered a conserved codon-bias axis. A/T-ending codons consistently marked genes involved in proliferation and RNA processing, whereas G/C-ending codons were enriched for differentiation and neuronal functions. While GC3 scores, measuring the third-nucleotide codon bias, showed differences between humans and rodents due to recombination events, the functional dichotomy was conserved. Isoacceptors frequencies, measuring gene synonymous codon bias, was conserved from rodents to humans. Synonymous codons exhibited distinct functional enrichment patterns, demonstrating functional divergence at the codon level. Two new indices; the ANN-index and mG-index, reflecting codons decoded by the tA and mG tRNA modifications, linked tRNA modification biology to translation. Both indices correlated with proliferative, A/T-biased programs, providing a universal basis for their roles in cancer. Tissue proteomes showed strong RNA-protein discordance and distinct codon biases. Analysis of 21 cancer types revealed a global A/T-ending codon bias in cancer. Analysis of 2,600 cancer cell lines revealed codon bias heterogeneity in cell lines from the same cancer subtype that is not observable between cancer patients. ConclusionsOur results define synonymous codon divergence and tRNA-modification indices as determinants of translational reprogramming. This work establishes a unified framework connecting codon usage, tRNA modifications, and proteome remodeling, providing a basis for rational design of mRNA and gene therapeutics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/685942v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@161ca0borg.highwire.dtl.DTLVardef@117c3f8org.highwire.dtl.DTLVardef@142d26borg.highwire.dtl.DTLVardef@483f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Xenophagocytosis blockade enhances interspecies chimerism

Organ shortage remains a major challenge in transplantation medicine. Interspecies blastocyst complementation is a promising approach to generate human organs in livestock hosts. However, getting xenogeneic donor cells to engraft and expand at early stages remains challenging. Here we identify an innate immune barrier, wherein host macrophages selectively recognize and eliminate viable xenogeneic donor cells. These events represent a form of phagoptosis and highlight a xenogeneic clearance process that we term xenophagocytosis. We identify the mechanism by which host macrophages selectively phagocytize xenogeneic donor cells: xenogeneic cells display elevated phosphatidylserine, an "eat-me" signal recognized by host macrophages through phagocytic receptor Axl. Xenophagocytosis blockade improves both rat and human donor chimerism in mouse embryos, indicating a conserved mechanism. These findings reveal potential mechanisms by which innate immune cells eliminate xenogeneic cells in early embryogenesis to preserve species integrity and offer improved strategies for generating human organs in livestock.

immunology↗

bioRxiv2025

Temporal dynamics of mRNA translation dysregulation and codon decoding during murine stroke evolution.

Ischemic stroke imposes acute oxidative and metabolic stress, yet how this reshapes mRNA translation remains poorly defined. We generated a time-resolved RNA-seq and ribosome-profiling atlas of mouse ischemic cortex at 1, 6 and 24 hours. Stroke produced early discordance between mRNA abundance and ribosome occupancy, revealing translational efficiency changes not captured by transcriptomics. The hyperacute phase was marked by a transient shift toward G/C-ending codon usage and a burst of 3' untranslated region ribosome footprints consistent with stop-codon readthrough-like termination stress. Machine-learning analyses linked this response to stop-proximal ribosome queuing, 3' untranslated region frame usage, local RNA structure and transcript architecture rather. From 6 to 24 hours, ischemic cortex developed progressive A-site pausing and altered coding-frame occupancy. Hidden Markov modeling localized a subset of frame-disruption events to discrete coding-sequence windows and machine learning revealed reproducible codon, amino-acid, nucleotide-composition and RNA-structure features associated with frameshifting. ORF-level analyses showed that many frame changes coincided with differential open reading frame usage. Late injury was characterized by increased upstream open reading frame engagement and 5' untranslated region ribosome occupancy, inversely correlating with coding-sequence translation. These data identify staged translational remodeling after stroke that could not be captured via traditional mRNA or protein expression analysis.

molecular biology↗

bioRxiv2025

The RNA-Binding Protein HuR Regulates Splicing Programs in Osteocytes

Post-transcriptional gene regulation is central to maintaining cellular homeostasis, among its mechanism alternative splicing (AS) fine-tunes cellular adaptation to stress. In this study we employed an approach combining RNA splicing analysis to define RNA binding protein (RBP) motif enrichment around alternatively spliced exons in primary osteocytes cultured in high glucose conditions (HG). We identified the RBP human antigen R (HuR) as a top candidate regulator of AS. Loss of HuR reshaped the transcriptome through widespread changes in gene expression and splicing, converging on two major pathways: stress response and translational control. Functional validation of splicing revealed that HuR depletion heightened oxidative stress sensitivity and compromised cell viability under HG by stabilizing and upregulating TXNIP, a thioredoxin inhibitor. HuR knockdown also impaired mitochondrial mass and function and disrupted key translational signals, despite preserving global protein output. These findings establish HuR as a central post-transcriptional regulator of osteocyte survival and metabolic adaptation under high glucose stress, with potential implications for hyperglycemic bone fragility.

molecular biology↗

bioRxiv2025

Donor-Targeted Anti-HLA-A2 Antibody Shows Nanogram-Level Efficacy in a Novel GVHD Mouse Model

Acute graft-versus-host disease (GVHD) remains a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with limited treatment options for steroid-refractory cases. Current therapies broadly suppress immune responses, increasing infection risk and impairing graft function. We hypothesized that selective depletion of donor immune cells through HLA allele-specific targeting could offer a safer and more precise alternative. To test this, we developed a monoclonal antibody (AN7) against HLA-A2 by immunizing HLA-A24 transgenic mice with recombinant HLA-A2 tetramers, followed by hybridoma screening. AN7 specifically recognized HLA-A2 and HLA-A68, and was reformatted into murine IgG2a and human chimeric IgG1 recombinant antibodies. These retained binding specificity and mediated robust complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) against HLA-A2+ target cells. To evaluate therapeutic potential, we established a fully MHC-mismatched allo-HSCT model using HLA-A2 transgenic donor bone marrow in lethally irradiated BALB/c recipients. A single ultra-low dose of AN7 (1-100 ng) administered post-transplant significantly improved survival and reduced GVHD while preserving donor hematopoiesis. These findings highlight the potential of allele-specific antibody-mediated clearance as a mechanistically distinct approach to donor immune modulation in GVHD.

immunology↗

bioRxiv2025

Selective Biogenesis and Structural Diversity Shape tRNA-Derived Fragment Landscapes across Mouse Organs

Transfer RNA derived small RNAs (tDRs) are increasingly recognized as versatile regulators, yet their physiological landscape remains poorly charted. We re-analyzed a small-RNA-seq dataset generated with tDR-optimized library preparation from seven adult mouse tissues to explore tissue specific tDRs enrichment. We catalogued 26901 unique nuclear tDRs (ntDRs) and 5114 mitochondrial tDRs (mtDRs). Library sizes and subclass composition varied markedly: heart was mtDRs-rich, whereas spleen produced the fewest reads but the greatest diversity. Clustering analysis segregated tissues, with spleen and lung forming a distinct immune cluster. Tissue-versus-all differential analysis showed the spleen harboring unique ntDRs and mtDRs. The brain preferentially accumulated 5'tiRNAs, while other organs were enriched for i-tRFs or tRF3s. Enriched fragments in different tissues originated from specific isoacceptors and isodecoder tRNAs independent of mature tRNA abundance, implying selective biogenesis rather than bulk turnover. G-quadruplex prediction revealed pronounced enrichment of potentially quadruplex-forming ntDRs in the spleen, predominantly Gly-, Asp- and Val-derived i-tRFs/tRF3s, suggesting structure-dependent functions in immune regulation. Collectively, our atlas reveals extensive tissue-specific heterogeneity in tDR biogenesis, sequence and structure, providing a framework for deciphering context-dependent regulatory roles of tDRs.

molecular biology↗

bioRxiv2025

Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD+ and SIRT upregulation

Emerging evidence strongly supports a close relationship between age-related hearing loss and frailty, highlighting the importance of early detection and intervention. Recently, we invented a mitochondria-homing drug named mitochonic acid 5 (MA-5), that increases the adenosine triphosphate (ATP) levels, rescue mitochondrial function, and protect tissue damages. Currently, the phase I clinical trial has been finished in Japan (jRCT2031210495) and the phase 2 clinical trial has already been approved by PMDA. Here we show that MA-5 improved various types of hearing loss in mouse models. Structural chemical bioanalysis revealed that MA-5 is a mixture of equal amount of S- and R- enantiomer and both S- and R- enantiomer increase ATP by binding mitochondrial protein, mitofilin. However, S-enantiomer significantly increased the NAD+ levels by binding to the NAD+-producing key enzyme nicotinamide phosphoribosyltransferase (NAMPT). Moreover, the S-enantiomer increased the sirtuin 1 protein by suppressing polyubiquitination induced by tripartite motif containing 28 (TRIM28) phosphorylation which was triggered by DNA-dependent protein kinase (DNA-PK) activation in the absence of DNA damage. Transcriptomic signatures showed that the signature of MA-5 shows an inverse correlation with aging and mortality and is oriented in the same direction as the OSKM-related iPSCs, suggesting the modification of aging pathways. Oral administration of MA-5 to mitochondrial disease model mouse showed increased survival. Our findings suggest that, in addition to enhancing ATP levels, the coordinated regulation of NAD+ metabolism, SIRT protein expression, and DNA-PK activity-constituting a novel therapeutic triad may contribute to the amelioration of hearing impairment and mitochondrial dysfunction, thereby improving life prognosis.

cell biology↗

bioRxiv2025

Development of iPSC-Derived T Cells Targeting EGFR Neoantigens in Non-Small Cell Lung Cancer

A long-sought goal of cancer immunotherapy is to mass-produce T cells that specifically target tumor neoantigens. One decisive challenge is the identification of neoantigens derived from cancer driver genes. Here, we identify T cells that recognize the NSCLC-associated EGFR C797S mutation, which confers resistance to current inhibitors and is linked to poor prognosis. To overcome limitations in T cell availability, we reprogrammed EGFR C797S-specific T cells into induced pluripotent stem cells (iPSCs) and re-differentiated them into CD8 T cells. These iPSC-derived T cells specifically recognized the EGFR C797S mutation and effectively killed cancer cells expressing this mutation. Our findings underscore the potential of targeting driver mutation-derived neoantigens for immunotherapy and demonstrate that iPSC-derived T cells can mediate antitumor effects. Collectively, this approach combining neoantigen identification with T cell reprogramming may offer a promising strategy for targeting drug-resistant tumors.

immunology↗

bioRxiv2025

Endothelial Sphingosine-1-Phosphate Receptor (S1PR) 1, a Functional S1PR in the Human Cerebrovascular Endothelium, Limits Blood Brain Barrier Permeability and Neuronal Injury following Subarachnoid Hemorrhage in Mice.

Hypoxia-induced blood-brain barrier (BBB) permeability has been identified as a key contributor to the progression of ischemic-hypoxic brain injury and neuronal dysfunction in stroke and other cerebrovascular diseases. Emerging clinical evidence highlights that the vasoprotective signaling properties of high-density lipoprotein (HDL), mediated through its bioactive lipid component sphingosine-1-phosphate (S1P), may be impaired in cardiovascular and inflammatory conditions. Nonetheless, the precise contributions and mechanistic roles of S1P signaling within the cerebral microvasculature remain insufficiently characterized. In this study, we aimed to elucidate the role of S1P signaling via its endothelial receptor S1PR1 in the pathophysiology of early brain injury following subarachnoid hemorrhage (SAH), a particularly severe form of stroke. Additionally, we sought to evaluate the relevance of the endothelial S1PR1 pathway in the human cerebrovascular endothelium, its functional role in hypoxia-induced cerebral endothelial barrier dysfunction, and its underlying molecular mechanisms. To address these objectives, we utilized endothelial-specific S1PR1 knockout mice subjected to the endovascular rupture model of aneurysmal SAH, performed mechanistic studies in primary human cerebral microvascular endothelial cells, and characterized S1PR1 expression in human brain tissue using validated protocols. Our findings reveal robust expression of S1PR1 in the cerebrovascular endothelium of both mice and humans. Functional analyses demonstrated that S1PR1 is critical for maintaining BBB integrity and mitigating neuronal injury in the context of SAH. Mechanistic in vitro studies indicated that S1PR1 exerts a vasoprotective effect by limiting hypoxia-induced BBB dysfunction in human primary brain microvascular endothelial cells through inhibition of Rho-associated kinase (ROCK)-mediated phosphorylation of myosin light chain (MLC), suppression of stress fiber formation and caveolin-1-dependent endosomal trafficking. These results highlight the pivotal role of endothelial S1PR1 signaling in preserving cerebral vascular integrity and provide a strong scientific foundation for developing novel therapeutic approaches targeting the S1P pathway in the endothelium to enhance neurovascular protection.

neuroscience↗

bioRxiv2024

Transcriptome-wide alternative mRNA splicing analysis reveals post-transcriptional regulation of neuronal differentiation.

Alternative splicing (AS) plays important roles in neuronal development, function, and diseases. Efforts to analyze AS transcriptome-wide in neurons remain limited. We characterized the transcriptome-wide AS changes in SH-SY5Y neuronal differentiation model, which is widely used to study neuronal function and disorders. Our analysis revealed global changes in five AS programs that drive neuronal differentiation. Motif analysis revealed the contribution of RNA binding proteins (RBPs) to the regulation of AS during neuronal development. We focused on the predominant AS program during differentiation, exon skipping (SE) events. Motif analysis revealed motifs for PTB and HuR/ELAVL1 to be the top enriched in SE events, and their protein levels were downregulated after differentiation. shRNA Knockdown of either PTB and HuR were associated with enhanced neuronal differentiation and transcriptome-wide exon skipping events driving the process of differentiation. At the level of gene expression, we observed only modest changes, indicating predominant post-transcriptional effects of PTB and HuR. We also observed that both RBPs altered cellular responses to oxidative stress, in line with the differentiated phenotype observed after KD. Our work characterizes the AS changes in a widely used and important model of neuronal development and neuroscience research and reveals intricate post-transcriptional regulation of neuronal differentiation.

neuroscience↗

bioRxiv2024

Angiogenin regulates mitochondrial stress and function via tRNA-derived fragments generation and impacting tRNA modifications.

Mitochondrial stress and dysfunction play an important role in many diseases, such as cancer, diabetes, and neurodegenerative diseases. We previously observed that mitochondrial electron transport chain (ETC) inhibition can induce tRNA cleavage and tsRNAs (tRNA-derived small non-coding RNAs) generation. However, whether this process is mediated via Angiogenin (ANG), the canonical enzyme responsible for tRNA cleavage, and whether it has a role in regulating the mitochondrial stress response remains to be understood. ANG is linked to Amyotrophic Lateral Sclerosis (ALS) and other conditions where mitochondrial stress plays a role in pathophysiology. Here, we aimed to examine the role of ANG in regulating the translational response to mitochondrial stress. We observed that ANG protected the cells from respiratory complex III and V inhibition specifically. Furthermore, we validated that the tsRNAs generated during mitochondrial and oxidative stress are mediated by ANG, given that their production is abrogated after ANG knock-out (KO). In addition, we observed that ANG-KO altered the tRNA modification status. Namely, we observed that ANG-KO led to the downregulation of queuosine tRNA modifications (tRNA-Q). tRNA-Q itself is related to mitochondrial translation and function. Indeed, we observed that ANG-KO led to reduced mitochondrial respiration and function. ANG altered how the cells respond to mitochondrial stress by altering the dynamic tRNA modification changes occurring during the stress response. We further examined the impact of ANG-KO on stress granules (SG) assembly as well as the knockdown of G3BP1 (core protein of SGs) on tsRNAs generation. Our results indicate that ANG regulates mitochondrial function and stress via tsRNAs generation as well as altering tRNA modifications levels. Our data also indicate that there are no direct links between tRNA cleavage and SG assembly, and both could be parallel systems for translation repression during stress.

molecular biology↗

bioRxiv2024

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

molecular biology↗

bioRxiv2023

Mammalian tissue specific translation regulation; role of tRNA epitranscriptome in regulating codon optimality patterns across tissues.

The tRNA epitranscriptome has been recognized as an important player in mRNA translation regulation. Our knowledge of the role of tRNA epitranscriptome in fine-tuning translation codon decoding at tissue or cell levels remains incomplete. Here, we analyzed seven tissues from mice for the expression of tRNA modifications and mature tRNAs as well as mRNA translation and codon decoding. Our analysis revealed distinct enrichment patterns of tRNA modifications in tissues. Queuosine (Q) tRNA modification was most enriched in the brain compared to other tissues, while mitochondrial tRNA modifications and tRNA expression was highest in the heart. Using three different metrics for codon analysis; isoacceptors frequencies, total codon frequencies, and A-site pausing, we revealed a strong bias towards A/T ending codons in most tissues except for the brain. Using this observation, we synthesized, and delivered in vivo, codon mutated EGFP for Q-codons, where the C-ending Q-codons were replaced with U-ending codons. The protein levels of mutant EGFP were downregulated in liver, which is poor in Q, when NAC codons were exchanged for NAU codons, while in brain EGFP levels did not change. This data shows that understanding tRNA modifications enrichments across tissues is not only essential for understanding codon decoding and bias, but it can also be utilized for optimizing gene and mRNA therapeutics to be more tissue, cell, or condition specific.

molecular biology↗

bioRxiv2023

Dynamic mRNA stability changes buffer transcriptional activation during neuronal differentiation and are regulated by RNA binding proteins.

The steady state levels of mRNA are outcomes of a finely tuned interplay between RNA transcription and decay. Therefore, the modulation of RNA stability is generally assumed to influence RNA abundance in a positive direction. However, the correlation between mRNA transcription, translation and stability remains elusive. Here, we employed a newly developed simplified mRNA stability profiling technique to explore the role of mRNA stability in SH-SY5Y neuronal differentiation model. Transcriptome-wide mRNA stability analysis revealed neural-specific RNA stability kinetics, including stabilization of transcripts encoding regulators of neuronal morphogenesis and function and destabilization of mitochondrial electron transport and redox homeostasis. When we further examined the relationship between transcription, translation and mRNA stability, a bidirectional regulation of RNA stability was revealed, wherein mRNA stability could either exert the buffering effect on gene products or change in a same direction as transcription. Motif analysis unveiled SAMD4A as a major regulator of the dynamic changes in mRNA stability observed during differentiation. Knockdown of SAMD4A impaired neuronal differentiation and influenced the response to oxidative stress. Mechanistically, SAMD4A was found to alter the stability of several mRNAs to which it binds. Meanwhile, a dimorphic pattern of the correlation between gene expression and SAMD4A-regulated mRNA stability was observed, suggesting dynamic regulation mRNA stability during the neuronal differentiation guided by SAMD4A. The novel insights into the interplay between mRNA stability and cellular behaviors provide a foundation for understanding neurodevelopmental processes and neurodegenerative disorders and highlights dynamic mRNA stability as an important layer of gene expression regulation.

molecular biology↗