Biology subjects

Nojima, T.

Publications and source records attributed to Nojima, T..

13 recordsLinked to original sources

bioRxiv2025

Meat the Fat: From High Lipid Content to Nutritional Enhancement to Reconstruct Wagyu Fat with Tunable Rheology and Organoleptic Fidelity for Cultivated Meat

Cultivated meat holds promise as a sustainable and ethical alternative to conventional meat, but reproducing the rich flavor, texture, and lipid composition of premium beef, such as Wagyu, remains a major hurdle. In this study, we redefine bovine adipogenesis by systematically optimizing edible biomaterials, fatty acid delivery systems, and culture conditions to engineer high-fat cultivated tissues with authentic sensory properties. Among various candidates, 1% alginate emerged as the optimal scaffold, providing mechanical softness compatible with adipogenic differentiation and mimicking the rheological properties of native fat. Supplementation with oleic acid complexed to edible albumins, especially whey protein (WP), significantly enhanced lipid accumulation and monounsaturated fatty acid content, particularly oleic acid, which reached up to 80.6% of total fatty acids. Engineered fat tissues displayed soft, spreadable textures and organoleptic characteristics consistent with high-quality beef fat. Fatty acid profiling revealed a bovine-like omega-6 signature, dominated by linoleic, arachidonic, and dihomo-{gamma}-linolenic acids, alongside detectable omega-3 subtypes including alpha-linolenic acid and trace levels of EPA and DHA. This composition not only validates the physiological relevance of the engineered fat, but also highlights its potential to deliver bioactive lipids rarely found in terrestrial animal products. Additionally, the WP-oleic acid system supported robust adipogenesis even under lower serum conditions. These findings establish a food-grade, tunable platform for producing physiologically and nutritionally enhanced fat tissues, advancing the realism, functionality, and health potential of next-generation cultivated meat.

bioengineering↗

bioRxiv2025

SETD2 methyltransferase activity aids gene definition by promoting correct transcription initiation and termination

SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma (RCC). In RCC, SETD2 mutations have been linked to delayed transcription termination, but not explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. Majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15-25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity, however widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3' pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and unrelated to transcription readthrough. In summary, we demonstrate that methyltransferase activity of SETD2 regulates transcriptional integrity by stimulating proper initiation, preventing cryptic initiation and promoting efficient 3' end processing.

molecular biology↗

bioRxiv2025

A High-Resolution Atlas of the Brain Predicts Lineage and Birth Order Underly Neuronal Identity.

Gene expression shapes the nervous system at every biological level, from molecular and cellular processes defining neuronal identity and function to systems-level wiring and circuit dynamics underlying behaviour. Here, we generate the first high-resolution, single-cell transcriptomic atlas of the adult Drosophila melanogaster central brain by integrating multiple datasets, achieving an unprecedented tenfold coverage of every neuron in this complex tissue. We show that a neurons genetic identity overwhelmingly reflects its developmental origin, preserving a genetic address based on both lineage and birth order. We reveal foundational rules linking neurogenesis to transcriptional identity and provide a framework for systematically defining neuronal types. This atlas provides a powerful resource for mapping the cellular substrates of behaviour by integrating annotations of hemilineage, cell types/subtypes and molecular signatures of underlying physiological properties. It lays the groundwork for a long-sought bridge between developmental processes and the functional circuits that give rise to behaviour.

neuroscience↗

bioRxiv2025

A Role for Exaptation in Sculpting Sexually Dimorphic Brains from Shared Neural Lineages.

Sex differences in behaviours arise from variations in female and male nervous systems, yet the cellular and molecular bases of these differences remain poorly defined. Here, we take an unbiased, single-cell transcriptomic approach to uncover how sex shapes the adult Drosophila melanogaster brain. We show that sex differences do not result from large-scale transcriptional reprogramming but through fine-tuning of otherwise shared developmental templates via the sex-differentiating transcription factors Doublesex and Fruitless. We reveal, with unprecedented resolution, the extraordinary genetic diversity within these sexually dimorphic cell types and find birth order represents a novel axis of sexual differentiation. Neuronal identity in the adult reflects spatiotemporal patterning and sex-specific survival, with female-biased neurons arising early and male-biased neurons arising late. This pattern reframes dimorphic neurons as "paralogous" rather than "orthologous", suggesting sex leverages distinct developmental windows to build behavioural circuits and highlights a role for exaptation in diversifying the brain.

neuroscience↗

bioRxiv2025

Single-molecule multimodal timing of in vivo mRNA synthesis

mRNA synthesis requires extensive pre-mRNA maturation, the organisation of which remains unclear. Here, we directly sequence pre-mRNA without metabolic labelling or amplification to resolve transcription and multimodal pre-mRNA processing at single-molecule resolution. Using poly(A) tail measurement, we distinguish transcriptionally engaged pre-mRNA from polyadenylated transcripts, revealing that splicing is substantially delayed compared to previous estimates. Splicing is rare for at least 10 kb behind elongating RNA polymerase II such that thousands of genes remain largely unspliced during transcription, revising the notion that splicing is predominantly co-transcriptional. Progressive splicing becomes apparent only on polyadenylated transcripts, suggesting that splicing is commonly activated after 3' end formation. Unexpectedly, we find abundant m6A on unspliced pre-mRNA, indicating that a substantial portion of RNA methylation precedes splicing. This m6A methylation is suppressed around exon boundaries, indicating that m6A topology is established prior to exon junction complex deposition. Finally, we demonstrate that 3' end cleavage occurs multiple kilobases behind transcription in coordination with efficient transcription termination, and further highlight recursive 3' end formation across hundreds of genes. Conserved from human cells to mouse tissues, we illustrate a revised timeline for mRNA synthesis wherein cleavage, termination, and m6A deposition occur earlier than, or at least partially decoupled from, the bulk of splicing, reframing the sequence of early mammalian gene expression.

genomics↗

bioRxiv2024

DNA-directed termination of mammalian RNA polymerase II

The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage of the nascent RNA. The RNAPII-associated cleavage product is then degraded by XRN2, dislodging RNAPII from the DNA template. In contrast, prokaryotic RNAP and eukaryotic RNAPIII often terminate directly at T-tracts in the coding DNA strand. Here, we demonstrate a similar and omnipresent capability for mammalian RNAPII. XRN2- and T-tract-dependent termination are independent - the latter usually acting when XRN2 cannot be engaged. We show that T-tracts terminate snRNA transcription, previously thought to require the Integrator complex. Importantly, we find genome-wide termination at T-tracts in promoter-proximal regions, but not within protein-coding gene bodies. XRN2-dependent termination dominates downstream of protein-coding genes, but the T-tract process is sometimes employed. Overall, we demonstrate global DNA-directed attrition of RNAPII transcription, suggesting that RNAPs retain the potential to terminate over T-rich sequences throughout evolution.

molecular biology↗

bioRxiv2024

Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations.

Classical Anfinsen's dogma states that a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, recent advances have revealed that single amino acid sequences can fold into two or more conformations. Here, we propose a novel approach to design a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal {beta}-strand and DHFR lacking the N-terminal {beta}-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analysis, including AFM imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. In addition, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a new direction for creating artificial proteins with on-off behaviors.

biochemistry↗

bioRxiv2024

NELF coordinates Pol II transcription termination and DNA replication initiation

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD, a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTSO_LIExpression of NELFCD transcript is up-regulated in colorectal tumors C_LIO_LINELF-C protein is mandatory for the transition between G1-S phases during cell cycle C_LIO_LINELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausing C_LIO_LINELF-C loss leads Pol II to invade DNA replication initiation zones C_LI

molecular biology↗

bioRxiv2023

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3UTR (3UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3UTR-APA and IPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3RNA-Seq were performed to quantify gene expression and characterize new 3UTR-APA and IPA mRNA isoforms. Our results show that polarization of human macrophages from naive to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3UTR and in IPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3UTR-APA and IPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induce 3UTR-APA alterations. Notably, some of these gene expression differences were also found in tumour-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro-inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 3UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools.

molecular biology↗

bioRxiv2021

CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

AbstractCDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

molecular biology↗

bioRxiv2020

Point Technology Illuminates The Processing Of Polymerase-Associated Intact Nascent Transcripts

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing. HIGHLIGHTSO_LIPOINT methodology dissects intact nascent RNA processing C_LIO_LISpecificity of Xrn2 exonuclease in co-transcriptional RNA degradation C_LIO_LISplicing suppresses Xrn2-dependent premature termination C_LIO_LIDifferent kinetic classes of co-transcriptional splicing in human genes C_LI

genomics↗

bioRxiv2019

Enhancers predominantly regulate gene expression in vivo via transcription initiation.

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

genomics↗

bioRxiv2018

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3 mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

molecular biology↗