Biology subjects

Kato, M.

Publications and source records attributed to Kato, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Evaluation of graphene oxide-mediated NET formation using HL-60-derived neutrophil-like cells

Neutrophil extracellular traps (NETs) are chromatin-based structures released by activated neutrophils in response to pathogens or chemical stimuli, contributing to host defense but also implicated in autoimmune disease and inflammation. As NET formation gains attention as an endpoint in in vitro immunotoxicity screening, the lack of reproducible and scalable systems hampers its broader application. Here, we developed an in vitro assay using HL-60-derived neutrophil-like cells (dHL-60) differentiated with all-trans retinoic acid to evaluate NET-inducing activity in a standardized, non-animal model. Graphene oxide (GO), a model nanomaterial known to trigger NETs in primary neutrophils, induced concentration-dependent NET formation in dHL-60 cells, with maximal induction at intermediate doses and attenuation at higher concentrations, likely due to particle aggregation. NET formation was validated by extracellular DNA staining and scanning electron microscopy. GO also induced superoxide-mediated ROS production, as confirmed by electron spin resonance, consistent with canonical NETosis pathways. Furthermore, GO suppressed PMA-induced NET formation, suggesting a dose- and context-dependent immunomodulatory effect. Collectively, our results demonstrate that dHL-60 cells recapitulate key features of NETosis observed in primary neutrophils and provide a practical, reproducible model for investigating immune responses to nanomaterials. This system supports the development of non-animal approaches for assessing immunological effects of chemical substances under controlled in vitro conditions.

pharmacology and toxicology↗

bioRxiv2025

A genome-wide, machine learning-guided exploration of the cis-regulatory code involved in neuronal differentiation

Gene expression is controlled by proximal and distal cis-regulatory elements (CREs), containing DNA motifs bound by various transcription factors (TFs). Other sequence features, such as specific k-mers or low complexity regions, have also been implicated [1-3]. However, in a dynamic biological process such as cell differentiation, we lack an understanding of how the transcriptional activity of CREs progressively change and what sequence features underlie these transitions, which may reflect common and/or coordinated regulatory processes. Here, we use single-cell ATAC-seq and RNA-seq to follow, at a genome scale, CREs along differentiation of induced pluripotent stem cells into cortical neurons and develop a method to automatically identify the diversity of CRE profiles and their underlying sequence features. We propose a machine-learning guided clustering algorithm, STOIC (Statistical learning TO Inform Clustering), that jointly learns an unsupervised clustering of the CREs in the space of the activity profiles and a supervised predictor associated with each cluster in the DNA-sequence space. STOIC is specifically designed to provide readily interpretable results. We show that the method identifies CRE profiles associated with highly predictive sequence features and outperforms methods solely concerned with co-activity clustering on this task. Orthogonal data collected in the same settings link the inferred CRE clusters to specific enhancer or promoter signatures. Furthermore, we show that the DNA features unveiled by STOIC reflect biologically relevant regulators and offer a valuable basis to dissect elements of the cisregulatory grammar. Finally, we demonstrate the general applicability of STOIC by analyzing five bulk CAGE datasets of human cells responding to various treatments.

bioinformatics↗

bioRxiv2025

Neurosecretory protein GL stimulates feeding behavior and fat accumulation in Japanese quails (Coturnix japonica)

Neurosecretory protein GL (NPGL) is a novel hypothalamic neuropeptide that promotes fat accumulation in rats, mice, and neonatal chicks. However, its role in Japanese quails (Coturnix japonica) remains unclear. In this study, we investigated the effects of chronic intracerebroventricular infusion of NPGL on body mass, food intake, and fat accumulation in five-week-old male Japanese quails. A 13-day administration of NPGL significantly increased body mass, food intake, and the masses of subcutaneous fat, abdominal fat, and liver. In contrast, water intake and the masses of the pancreas, testes, heart, and muscle remained unchanged. Blood concentrations of triglyceride, glucose, and non-esterified fatty acid were unaffected. Real-time qPCR analysis revealed a significant upregulation of NPGM, a paralogous gene of NPGL, in the hypothalamus. Additionally, the expression of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in lipogenesis, showed an increasing trend in the liver. Although the fatty acid ratio did not change in the SCD1 activity index (palmitoleate/palmitate), the de novo lipogenesis index (palmitate/linoleate) exhibited an upward trend in both the liver and abdominal fat. These results suggest that NPGL promotes fat accumulation in Japanese quails, highlighting its potential role as a contributing factor to obesity in avian species.

zoology↗

bioRxiv2025

Cell type-specific intronic RNAs shape genome architecture during neuronal lineage specification

Cell differentiation towards neurons is accompanied by widespread changes in three-dimensional (3D) genome organization and gene expression. Chromatin-associated RNAs have been proposed to be important regulators of such changes; however, the type, abundance, and role of these RNAs during neuronal differentiation remain largely unexplored. Here, we integrate multi-omic data generated in the frame of the Functional ANnoTation Of the Mammalian genome (FANTOM6) to chart 3D genome, RNA-DNA contactome, and transcriptome changes occurring during in vitro differentiation of human induced pluripotent stem cells to neural stem cells and neurons. We reveal a previously unreported phenomenon, in which intronic RNAs engage in long-distance contacts with DNA loci distributed all over the genome. These trans-contacting intronic RNAs (TIRs) are produced from exceptionally long (mean length: 750 kilobases, kb) protein-coding genes that carry ultra-long introns and are selectively expressed in neurons. We show that TIRs do not undergo rapid co-transcriptional degradation but rather accumulate in the nucleus of neuronal cells, forming large dot clouds around their source loci and spreading across the nucleus, as visualized by single-molecule RNA fluorescence in situ hybridization. TIRs engage in contacts with a set of genomic regions (TIR-contacted regions or TIRCs) that carry much shorter (mean length: [~]30 kb) neuronally expressed genes forming high-connectivity hubs. We also show that the expression of genes within TIRCs contacted by the same set of TIRs is highly co-varied, and that TIR source genes, especially their introns, are enriched in genetic risk loci for neurodevelopmental and neuropsychiatric disorders. Our findings point to a functional role of the persistence of long intronic RNAs in the nucleus of neuronal cells and might contribute to explain why neurons uniquely express many ultra-long genes. We propose a model in which TIRs form pan-nuclear scaffolds--which we propose to name introsomes--that constitute a dynamically self-renewing regulatory layer, where transcription itself continuously regenerates the very scaffold that organizes genome function in neurons, and that might be involved in the pathogenesis of neurodevelopmental and neuropsychiatric disorders.

genomics↗

bioRxiv2024

ER sensing of lipid metabolism drives PRA family-dependent regulation of COPII vesicle transport

Newly synthesized secretory proteins and many lipids are transported from the endoplasmic reticulum (ER) to the Golgi prior to their ultimate destinations. The ER-to-Golgi transport must be tightly regulated during adaptation to environmental stress. However, the sensing mechanism and regulatory pathways governing the consecutive formation, budding and transportation of COPII vesicles from the ER remain insufficiently explored. Here, we present evidence indicating that COPII-mediated vesicle transport is transcriptionally controlled through the phosphatidic acid-dependent Opi1-Ino2/Ino4 regulatory circuit. Our analysis indicates that YIP3, a target gene of Ino2/Ino4, exerts a negative regulatory impact on COPII-mediated vesicle transport. Furthermore, we demonstrated that Ino2/Ino4 but not Yip3 modulates Sar1 activation, the initial step in COPII vesicle formation, whereas Yip3 hinders Sec16 assembly on the ER membrane, thereby implying that Ino2/Ino4 governs COPII-mediated trafficking at multiple steps. Thus, this study provides the first evidence for an ER sensing system that transcriptionally fine-tunes multiple steps of anterograde vesicular transport in response to alterations in lipid composition of the ER membrane.

cell biology↗

bioRxiv2024

CFC-seq: identification of full-length capped RNAs unveil enhancer-derived transcription

Long-read sequencing has transformed transcriptome profiling, yet capturing full-length, non-polyadenylated transcripts like enhancer RNAs (eRNAs) remains challenging. Here, we introduce CFC-seq, combining cap-trapping and in vitro poly(A)-tailing to sequence poly(A) and non-poly(A) RNAs with precise transcription start site. Paired with our assembler, SALA, we identified 39,425 novel transcriptional units, including [~]24,000 eRNAs. Our data reveal a distinct genomic code governing eRNA fate dictated by core promoter architecture. CpG-island enhancers show high chromatin connectivity but yield short, exosome-sensitive RNAs. Conversely, TATA-box enhancers systematically co-opt LTR retrotransposons to inherit structural motifs that produce long, stable, and spliced RNAs. Mechanistically, the pioneer factor NF-Y activates these viral elements to license transcription, balanced by TEAD4 activity across a dual-gear regulatory axis. Finally, non-poly(A) eRNAs terminate via exosome-associated processing at structural-depleted cleavage zones. This comprehensive annotation links enhancer sequence architecture to RNA fate, providing a new transformative framework for decoding the functional human genome. HighlightsO_LIExpanded genomic architecture: CFC-seq unmasks a hidden layer of human transcriptome, identifying 39,425 novel transcriptional units with high-confidence TSS support, including [~]24,000 eRNAs. C_LIO_LITSS-first assembler: We introduce SALA, a specialized long-read assembler that prioritizes authentic 5 Cap-trapped ends to accurately reconstruct the TSS-resolved transcript models. C_LIO_LIGenomic code of eRNA fate: CGI enhancers drive short and exosome-sensitive transcripts associated with repressive H3K27me3 mark and high chromatin connectivity. TATA-box enhancers produce cell-type-specific, long, stable, and frequently spliced eRNAs. C_LIO_LIEvolutionary co-option of retrotransposons: A major fraction of TATA-box eRNAs originate from LTR retrotransposons, providing a direct mechanism for integration of viral elements into the human regulatory landscape. C_LIO_LIA dual-gear pioneering axis: The pioneer factor NF-Y activates unprimed LTR-TATA enhancers to license transcription independent of histone acetylation cascades, operating in parallel with TEAD4-mediated activation. C_LIO_LIStructural determinants of eRNA termination: Non-poly(A) eRNA TES features a secondary structure depletion zone that coordinates pol II termination and calibrates exosome-mediated turnover. C_LI

genomics↗

bioRxiv2024

Mutagenesis Sensitivity Mapping of HumanEnhancers In Vivo

Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo. Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic.

genomics↗

bioRxiv2024

RADIP technology comprehensively identifies H3K27me3-mediated RNA-chromatin interactions

Many RNAs associate with chromatin, either directly or indirectly. Several technologies for mapping regions where RNAs interact across the genome have been developed to investigate the function of these RNAs. Obtaining information on the proteins involved in these RNA-chromatin interactions is critical for further analysis. Here, we developed RADIP (RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation), a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA-chromatin interactions mediated by individual proteins. Building upon the foundational principles of RADICL-seq, RADIP extends its advantages by increasing genomic coverage and unique mapping rate efficiency compared to existing methods. To demonstrate its effectiveness, we applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells (mESCs). We identified a multitude of RNAs, including RNAs from protein-coding genes and non-coding RNAs, that are associated with chromatin via H3K27me3 and that likely facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome, thereby affecting gene expression, chromatin structures and pluripotency of mESCs. Our study demonstrates the applicability of RADIP to investigations of the functions of chromatin-associated RNAs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/597497v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@c41e0forg.highwire.dtl.DTLVardef@1919e78org.highwire.dtl.DTLVardef@13adc29org.highwire.dtl.DTLVardef@14c3db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Cryo-EM and Solid State NMR Together Provide a More Comprehensive Structural Investigation of Protein Fibrils

The Tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure. SignificanceThe use of multiple techniques to structurally characterize proteins provides models that accurately describe molecular conformations better than a technique used in isolation. Combination approaches allow for the study of proteins not only as rigid objects, but rather dynamic molecules that "breathe" over time. Cryogenic electron microscopy and solid state nuclear magnetic resonance are used jointly to provide a more detailed model of the same protein fibrils, and each technique provides novel insights.

biophysics↗

bioRxiv2024

Massively parallel reporter assays and mouse transgenic assays provide complementary information about neuronal enhancer activity

Genetic studies find hundreds of thousands of noncoding variants associated with psychiatric disorders. Massively parallel reporter assays (MPRAs) and in vivo transgenic mouse assays can be used to assay the impact of these variants. However, the relevance of MPRAs to in vivo function is unknown and transgenic assays suffer from low throughput. Here, we studied the utility of combining the two assays to study the impact of non-coding variants. We carried out an MPRA on over 50,000 sequences derived from enhancers validated in transgenic mouse assays and from multiple fetal neuronal ATAC-seq datasets. We also tested over 20,000 variants, including synthetic mutations in highly active neuronal enhancers and 177 common variants associated with psychiatric disorders. Variants with a high impact on MPRA activity were further tested in mice. We found a strong and specific correlation between MPRA and mouse neuronal enhancer activity including changes in neuronal enhancer activity in mouse embryos for variants with strong MPRA effects. Mouse assays also revealed pleiotropic variant effects that could not be observed in MPRA. Our work provides a large catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays.

genomics↗

bioRxiv2024

CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109 /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 "HIV-1 RNA copy number quantification" (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene of in vitro transcribed RNA molecules at low ({approx} 103 /L) and high concentration ({approx} 109 /L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808 /L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

molecular biology↗

bioRxiv2023

Bi-allelic variants in WDR47 lead to neuronal loss causing a rare neurodevelopmental syndrome with corpus callosum dysgenesis in humans.

The corpus callosum (CC) is the largest interhemispheric connection that is largely formed by the axons of layer 2/3 callosal projection neurons (CPNs) through a series of tightly regulated cellular events, including neuronal specification, migration, axon extension and branching. Defects in any of those steps may prevent the proper development of the corpus callosum resulting in a spectrum of disorders collectively referred to as corpus callosum dysgenesis (CCD). Here, we report four unrelated families carrying bi-allelic variants in WDR47 presenting with CCD together with other neuroanatomical phenotypes such as microcephaly, cerebellar abnormalities and hydrocephalus. Using a combination of in vitro and in vivo mouse models and complementation assays, we show that independently from its previously identified functions in neuronal migration and axonal extension, WDR47 is required for survival of callosal neurons by contributing to the maintenance of mitochondrial and microtubule homeostasis. We further provide evidence that severity of the CCD phenotype is determined by the degree of the loss of function caused by the human variants. Taken together, we identify WDR47 as a causative gene of a new neurodevelopmental syndrome characterized by corpus callosum abnormalities and other neuroanatomical malformations.

neuroscience↗

bioRxiv2023

Expression of mRNAs Encoding Hypothalamic Small Proteins, Neurosecretory Protein GL and Neurosecretory Protein GM, in the Japanese Quail, Coturnix japonica

Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are novel neuropeptides that have been discovered in the hypothalamic infundibulum of chickens. NPGL and NPGM play important roles in lipid metabolism in juvenile chickens. The physiological functions of NPGL and NPGM in sexually mature birds remain unknown. The Japanese quail (Coturnix japonica) seems to be an appropriate model for analyzing NPGL and NPGM during sexual maturity. However, studies on NPGL or NPGM have yet to be reported in the Japanese quail. In the present study, we identified cDNAs encoding precursor proteins of NPGL and NPGM in the quail hypothalamus. In situ hybridization revealed that NPGL mRNA-expressing cells in the hypothalamus were localized in the infundibular nucleus and median eminence, and NPGM mRNA-expressing cells were only found in the mammillary nucleus. Immunohistochemistry revealed that NPGM-like immunoreactive cells were distributed in the mammillary nucleus, whereas NPGL-like immunoreactive cells were not detected in the hypothalamus. Real-time PCR analysis indicated that the expression of NPGL mRNA was higher in the hypothalamus of females than in males, and NPGM mRNA expression showed no sex differences. NPGL and NPGM mRNA expression in males was upregulated after 24 h of food deprivation. In females, only NPGM mRNA expression was increased by fasting. These results suggest that the physiological functions of NPGL and NPGM are different in quail, and these factors are involved in sex differences in energy metabolism.

zoology↗

bioRxiv2023

Oxidative regulation of TDP-43 self-association by a beta-to-alpha conformational switch

An evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an -helical or {beta}-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-{beta} structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-{beta} structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of -helical structure in precisely the same region formerly functioning to facilitate {beta}-strand mediated self-association. We further observe that the -helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the {beta}-to- conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains. Significance StatementThe TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-{beta} structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an -helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.

biochemistry↗

bioRxiv2023

Cell Type- and Tissue-specific Enhancers in Craniofacial Development

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/546603v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@7595a9org.highwire.dtl.DTLVardef@1b48b81org.highwire.dtl.DTLVardef@32fe86org.highwire.dtl.DTLVardef@1e41fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Global changes in open reading frame dominance of RNAs during cancer initiation and progression

Cancer cells express unique RNA transcripts; however, the factors determining their translation have remained unclear. We recently developed open reading frame (ORF) dominance as a measure that correlates with coding potential of RNAs. Upon calculating the ORF dominance of cancer-specific transcripts across 24 human tumor types, 14 exhibited significantly higher ORF dominance in cancer than in normal tissues. In organoid-based mouse genetic models, ORF dominance increased with carcinogenesis. Gene ontology analysis revealed that gene sets with increased ORF dominance were associated with cell proliferation, while those with decreased ORF dominance were linked to DNA damage response. Translatome analyses demonstrated that elevated ORF dominance during carcinogenesis resulted in higher translation frequencies of ribosome-bound RNAs. As cancer progressed, ORF dominance showed that the boundary between coding and noncoding transcripts became blurred prior to distant metastasis, indicating decreased proliferative cell populations and increased generation of RNA isoforms that potentially translate neoantigens before the development of metastatic tumors. These findings suggest that cancer evolution leads to dynamic changes in ORF dominance, resulting in global translational alterations in transcriptomes.

cancer biology↗

bioRxiv2023

Abundant capped RNAs are derived from mRNA cleavage at 3'UTR G-Quadruplexes

The 3 untranslated region (3UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5 ends of mRNAs, additionally reveals a large number of apparently 5 capped RNAs derived from 3UTRs. Here we provide the first direct evidence that these 3UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. We also find that production of 3UTR-derived RNA could explain the previously reported role of a 3UTR G-quadruplex in regulating the production of APP protein. Taken together, we provide new insights into the origin and abundance of 3UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/538568v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3f2ca7org.highwire.dtl.DTLVardef@18ca1b6org.highwire.dtl.DTLVardef@1ccdd8dorg.highwire.dtl.DTLVardef@e58938_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Molecular Weight-dependent Diffusion, Biodistribution, and Clearance Behavior of Tetra-armed Poly(ethylene glycol) Subcutaneously Injected into the Back of Mice

Four-armed poly(ethylene glycol) (PEG)s are essential hydrophilic polymers extensively utilized to prepare PEG hydrogels, which are valuable tissue scaffolds. When hydrogels are used in vivo, they eventually dissociate due to the cleavage of the backbone structure. When the cleavage occurs at the cross-linking point, the hydrogel elutes as an original polymer unit, i.e., four-armed PEG. Although four-armed PEGs have been utilized as subcutaneously implanted biomaterials, the diffusion, biodistribution, and clearance behavior of four-armed PEG from the skin are essential. This paper investigates time-wise diffusion from the skin, biodistribution to distant organs, and clearance of fluorescence-labeled four-armed PEGs with molecular weight (Mw) ranging from 5-40 kg/mol subcutaneously injected into the back of mice. Changes over time indicated that the fate of subcutaneously injected PEGs is Mw-dependent. Four-armed PEGs with Mw [≤] 10 kg/mol gradually diffused to deep adipose tissue beneath the injection site and distributed dominantly to distant organs, such as the kidney. PEGs with Mw [≥] 20 kg/mol stagnated in the skin and deep adipose tissue, and were mainly delivered to the heart, lung, and liver. The fundamental understanding of the Mw-dependent behavior of four-armed PEGs is beneficial for preparing biomaterials using PEGs, providing a reference in the field of tissue engineering.

bioengineering↗