Biology subjects

Su, T.

Publications and source records attributed to Su, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Multiomics Reveals IGF-1 c.258A > G Reduces Triacylglycerol Containing Medium-Chain Fatty Acid in Milk

Fatty acid composition of milk triacylglycerol (TAG) is important for neonatal growth. We previously identified a synonymous mutation widely distributed in human populations, IGF-1 c.258A > G, which decreased milk TAG content in gene-edited mice. So far, the acyl chain composition of affected TAGs, underlying molecular mechanism, and cross-species conservation remain unclear. Here, integrated transcriptomic and metabolomic analyses of mouse mammary glands revealed that this mutation downregulates IGF-1 expression and elevates lipoprotein lipase (LPL) activity, and potential associations between IGF-1 and peroxisome proliferator-activated receptor {gamma} (PPARG), fatty acid synthase (FASN) and lipin 1 (LPIN1). Association analysis in 295 Bama pigs carrying this gene polymorphism showed reduced milk lipid content in sows; further, correspondingly lower litter weight was significantly associated with the genotype. Integrated metabolomic profiling of mouse mammary glands and porcine milk demonstrated insufficient production of lauric and myristic acids. Accordingly, supplementing these two fatty acids restored TAG content. Mechanistically, IGF-1 promotes triacylglycerol-containing medium-chain fatty acid (MCFA-TAG) synthesis by activating the PPARG-FASN/LPIN1 axis, whereas increased LPL activity indirectly suppresses this axis through long-chain fatty acid accumulation. Together, our results reveal the molecular mechanism whereby this mutation affects milk lipid synthesis, providing insights into the regulation of MCFA-TAG synthesis.

molecular biology↗

bioRxiv2026

Deep Proteoform Sequencing with Top-Down Direct Mass Technology

Individual Ion Mass Spectrometry (I2MS) using Direct Mass Technology mode on an Orbitrap mass spectrometer (DMTm) increases sensitivity, resolution, and mass range for protein analysis. Here, we present an end-to-end workflow for deep proteoform sequencing using top-down mass spectrometry with DMTm. By assigning the charge of individual fragment ions and converting spectra from the m/z to the mass domain, DMTm resolves overlapping isotopic distributions that have limited conventional top-down mass spectrometry. Across different fragmentation modes on Orbitrap mass spectrometers, top-down DMTm significantly outperformed conventional top-down mass spectrometry methods. For a glycosylated 50.8 kDa antibody heavy chain, sequence coverage was greatly increased, from 27.5% to 83.3%, in 10 minutes of acquisition using a single fragmentation mode. Coverage of the middle 350 residues improved from 0% to >95%, demonstrating near-complete coverage of the difficult-to-characterize internal region of a large protein. The fragmentation patterns of DMTm were found to be complementary to conventional top-down, with higher internal coverage for DMTm and higher terminal coverage for conventional. Accordingly, aggregation of the data from the two modes further increased heavy chain sequence coverage to 90.2%. A new software platform, Proteoform Studio, provided optimized ion processing for improved sequence coverage and enabled real-time experimental monitoring as individual ions were accumulated. The platform automatically integrates conventional and DMTm data to provide the most comprehensive sequence coverage possible. Together, these advances enable substantially deeper proteoform sequencing and establish a straightforward, complete top-down DMTm workflow to confidently define proteoforms in biological systems and biotherapeutic development.

bioinformatics↗

bioRxiv2026

Targeted Analysis of >1500 Plasma Proteoforms via Individual Ion Mass Spectrometry

Plasma proteomics has long sought to accessibly sense human biology, precisely detect disease states, and advance diagnostics through clinical translation. In recent years, companies with defined panels such as Olink and Somascan have entered the field to complement bottom-up mass spectrometry (BUP). This study leverages a novel mass spectrometry platform to capture targeted proteoform information lost by mainline antibody-, aptamer-, and BUP-driven workflows. The Plasma Proteoform Assay (PPA) uses Individual Ion Mass Spectrometry (I{superscript 2}MS) to resolve mixtures of intact proteins presented by direct injection. Two proteoform panels, PPA 526 and PPA 1514, were defined from human plasma samples obtained from 81 individuals. The panels quantify 526 proteoforms derived from 59 genes and 1,514 proteoforms from 155 genes, respectively. Reproducibility for both panels showed coefficients of variation below 20% for most proteoforms (59-80%). PPA was benchmarked in studies including subjects with hepatic cirrhosis (N=30) and resilient agers carrying a SERPINE1 (PAI 1) mutation (N=27). PPA signatures distinguished SERPINE1 mutation carriers from affected individuals and provided sufficient resolution to discriminate among cirrhosis disease stages. In summary, we present PPA 526 and PPA 1514, the first scalable plasma proteoform panels capable of tracking hundreds to thousands of targets in a few minutes per sample.

biochemistry↗

bioRxiv2026

The tumour suppressor RBM5 activates the helicase DHX15 to regulate splicing

Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is elusive. Using in vivo spliceosome capture and cryogenic electron microscopy, we determined structures of precatalytic spliceosomes arrested by RBM5 immediately after U2 snRNP branchpoint recognition. Despite intron diversity, the U2-pre-mRNA duplex, branchpoint adenine, and downstream polypyrimidine tract are well-resolved. RBM5 binds the outer SF3B1 HEAT surface and performs dual functions: First, its helix-loop-helix motif and upstream zinc-finger domain sterically block tri-snRNP and Prp8 docking and prevent progression to pre-B and Bact complexes; Second, its G-patch activates DHX15 and places this DExH-box helicase on the pre-mRNA as it exits SF3B1, poised for branch helix unwinding. DHX15 binding to SF3B1 is facilitated by U2SURP/SR140, which engages SF3B1 near RBM5s helix-loop-helix. Functional assays confirm that disruption of the RBM5 interfaces with either DHX15 or SF3B1 inhibit exon repression. Mutations at these regulatory interfaces are common in cancer genomes and predicted to disrupt its regulation of apoptotic isoforms. Thus, RBM5 acts as a dual-action spliceosome gatekeeper that couples helicase activation with physical stalling to enforce tumour-suppressive alternative splicing programmes.

molecular biology↗

bioRxiv2025

Mass spectrometry-based proteomic and glycoproteomic drafts of fifteen human body fluids

The study of proteins and N-glycosylation in human body fluids (HBFs) is crucial for understanding both physiological and pathological states, offering valuable insights for disease diagnostics and monitoring. In this study, we present a comprehensive proteomics and glycoproteomics dataset featuring 15 types of precious HBFs (plasma (P), urine (U), cerebrospinal fluid (CSF), pleural fluid (PF), ascitic fluid (AF), synovial fluid (SF), pericardial fluid (PCF), peritoneal dialysis effluent (PDE), bile (B), gastric juice (GJ), seminal plasma (SP), saliva (SA), pancreatic juice (PJ), sweat (SW), and tear (T)). We also provide detailed protocols for protein processing specific to each type of HBF. The resulting peptides and enriched intact N-glycopeptides were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data independent acquisition (DIA) and combined electron transfer/higher-energy collisional dissociation and stepped collision energy/higher-energy collisional dissociation (EThcD-sceHCD) mode, respectively. The resulting spectra and searched files have been uploaded to the online repository iProX (www.iprox.cn) and made easily accessible (PXD068799) to facilitate further research into HBF proteomics and glycoproteomics.

systems biology↗

bioRxiv2025

Top-Down Individual Ion Mass Spectrometry Reveals 85-110 kDa Catenin Phospho-Proteoforms Regulated by Actomyosin Contractility

A central challenge in top-down proteomics is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of {beta}- and -catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on {beta}-catenin and 7 on -catenin, with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at -catenin S641 was constitutive, while other sites in the P-linker and actin-binding domains as well as {beta}-catenin S675 and S552 were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry. Our results support a "catenin phospho-code" model, wherein combinatorial phosphorylation patterns encode mechano-transductive signals regulating cell-cell adhesion. This work establishes top-down I2MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.

biochemistry↗

bioRxiv2025

Replication stress alters CENP-A nucleosome stability during S phase

1.The maintenance of centromere identity is essential for the proper segregation of chromosomes during cell division. Centromere identity is epigenetically specified by centromeric histone H3 variant CENP-A, and its retention during DNA replication is facilitated by HJURP chaperone. Replication stress disrupts replication fork progression and can negatively influence the interactions between histone chaperone network necessary for retention and deposition of parental and new histones, respectively. In this study we investigate the role of replication stress response on centromere inheritance. We define changes in centromere-associated proteins that govern stability of centromeric and canonical nucleosomes through proximity labeling coupled with affinity purification mass spectrometry. We identified that under replication stress, CENP-A-containing chromatin strongly enriches for SWI/SNF chromatin remodeling proteins ATRX. We show that depletion of ATRX and its associated histone H3.3 chaperone DAXX results in the loss CENP-A retention in S-phase and loss persists into the subsequent cell cycle. Altogether our findings provide insight into how replication stress negatively influences centromeric chromatin instability and delineates a function of DAXX-ATRX complex in maintaining centromere inheritance during DNA replication.

biochemistry↗

bioRxiv2025

Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry

Histone post-translational modifications (PTMs) often serve as distinct recognition sites for the recruitment of chromatin-associated proteins (CAPs) for epigenome regulation. While CAP-PTM interactions have been extensively studied using histone peptides, this cannot consider the regulatory potential of multi-site binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables controlled disassembly of intact CAP:nucleosome (CAP:nuc) complexes to provide a direct readout of the contained histone proteoforms. As proof of principle, we show the BPTF PHD-BD native tandem reader requires coincident H3K4me3K9acK14acK18ac for effective nucleosome engagement. We extend our approach to explore how the BRD4 (native BD1-BD2), DNMT3A-MPP8 (chimeric PWWP-CD), and PtSHL (native BAH-BD) tandem readers interact with endogenous nucleosomes. Each reveals distinct enrichment profiles: BRD4 favoring di- and tri-acetylated histone H4 proteoforms, whereas DNMT3A-MPP8 and PtSHL preferentially interact with hypermethylated H3 proteoforms. Of note the latter enriches combinatorial {H3K4me3K27me3} on the same histone tail in HeLa chromatin, and thus expands the potential biology of this widely studied bivalent signature. By directly characterizing CAP:nuc complex composition with combinatorial PTM information in a single readout, Nuc-MS serves as a new approach to discover the modifications driving binding, and therefore primary candidates to explore for structural biology and genomic studies. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/651740v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8815e9org.highwire.dtl.DTLVardef@5685b5org.highwire.dtl.DTLVardef@643881org.highwire.dtl.DTLVardef@1b9bc20_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFor TOC only.C_FLOATNO Synopsis. Nuc-MS provides the ability to control the disassembly of chromatin-associated protein-nucleosome complexes (CAP-nuc) and delineate the histone proteoforms driving tandem reader domain binding. C_FIG

biochemistry↗

bioRxiv2025

Next-Generation Protein Sequencing and individual ion mass spectrometry enable complementary analysis of interleukin-6

The vast complexity of the proteome currently overwhelms any single analytical technology in capturing the full spectrum of proteoform diversity. In this study, we evaluated the complementarity of two cutting-edge proteomic technologies--single-molecule protein sequencing and individual ion mass spectrometry--for analyzing recombinant human IL-6 (rhIL-6) at the amino acid, peptide, and intact proteoform levels. For single-molecule protein sequencing, we employ the recently released Platinum(R) instrument. Next-Generation Protein Sequencing (NGPS) on Platinum utilizes cycles of N-terminal amino acid recognizer binding and aminopeptidase cleavage to enable parallelized sequencing of single peptide molecules. We found that NGPS produces single amino acid coverage of multiple key regions of IL-6, including two peptides within helices A and C which harbor residues that reportedly impact IL-6 function. For top-down proteoform evaluation, we use individual ion mass spectrometry (I2MS), a highly parallelized orbitrap-based charge detection MS platform. Single ion detection of gas-phase fragmentation products (I2MS2) gives significant sequence coverage in key regions in IL-6, including two regions within helices B and D that are involved in IL-6 signaling. Together, these complementary technologies deliver a combined 52% sequence coverage, offering a more complete view of IL-6 structural and functional diversity than either technology alone. This study highlights the synergy of complementary protein detection methods to more comprehensively cover protein segments relevant to biological interactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/637157v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@82d72aorg.highwire.dtl.DTLVardef@1fc39f3org.highwire.dtl.DTLVardef@40a677org.highwire.dtl.DTLVardef@70f6c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Social dysfunction relates to altered default mode network functional integrity across neuropsychiatric disorders: A replication and generalization study

BackgroundSocial dysfunction is an early manifestation of neuropsychiatric disorders that may relate to altered Default Mode Network (DMN) integrity. This study aimed to replicate previous findings linking social dysfunction with diminished resting-state DMN functional connectivity and altered task-based DMN functional activation in response to emotional faces across schizophrenia (SZ), Alzheimers disease (AD), and healthy controls (HC), and to extend these findings to major depressive disorder (MDD). MethodsResting-state fMRI and task-based fMRI data on implicit facial emotional processing were acquired in an overlapping cohort (resting-state fMRI: N=167; SZ=32, MDD=44, AD=29, HC=62. Task-based fMRI: N=152; SZ=30, MDD=42, AD=26, HC=54). Additionally, mega-analyses (N=317 for resting-state fMRI; N=291 for task-based fMRI) of the current and a prior independent sample were conducted. Social dysfunction was indexed with the Social Functioning Scale (SFS) and the De Jong-Gierveld Loneliness (LON) scale. ResultsThe association between higher mean SFS+LON social dysfunction scores and diminished DMN connectivity within the dorsomedial prefrontal cortex across SZ/AD/HC participants was replicated, and extended to MDD patients. Similar observations within the dorsomedial and rostromedial prefrontal cortex were found in the mega-analysis. Associations between social dysfunction and DMN activation in response to sad and happy faces were not replicated or found in the mega-analysis. ConclusionsDiminished dorsomedial prefrontal cortex DMN connectivity emerged as a transdiagnostic neurobiological marker for social dysfunction, suggesting a potential treatment target for precision medicine approaches. DMN functional responses to emotional faces may not be a sensitive biomarker for social dysfunction.

neuroscience↗

bioRxiv2024

Proteolysis-targeting vaccines (PROTAVs) for robust tumor combination immunotherapy

Protein/peptide subunit vaccines are promising to promote the tumor therapeutic efficacy of immune checkpoint blockade (ICB). However, current protein/peptide vaccines elicit limited antitumor T cell responses, leading to suboptimal therapeutic efficacy. Here, we present proteolysis-targeting vaccines (PROTAVs) that facilitate antigen proteolytic processing and cross-presentation to potentiate T cell responses for robust ICB combination immunotherapy of melanoma. PROTAVs are modular conjugates of protein/peptide antigens, E3 ligase-binding ligands, and linkers. In antigen-presenting cells (APCs), PROTAVs bind to E3 ligases to rapidly ubiquitinate PROTAV antigens, facilitating antigen proteolytic processing by proteasome, and thereby promoting antigen cross-presentation to T cells and potentiating CD8+ T cell responses. We developed a melanoma PROTAV using a tandem peptide of trivalent melanoma-associated antigens. Co-delivered by lipid nanoparticles (LNPs) with bivalent immunostimulant adjuvants, this PROTAV promotes the quantity and quality of melanoma-specific CD8+ T cells in mice. Further, combining PROTAV and ICB ameliorates the immunosuppressive melanoma microenvironment. As a result, PROTAV and ICB combination enhances melanoma complete regression rates and eradicated 100% large BrafV600E melanoma without recurrence in syngeneic mice. PROTAVs hold the potential for robust tumor combination immunotherapy.

bioengineering↗

bioRxiv2024

α/β-Hydrolase domain-containing 6 (ABHD6) accelerates the desensitization and deactivation of TARP γ-2-containing AMPA receptors

AMPA receptors (AMPARs) mediate most of the fast excitatory synaptic transmission in the mammalian brain. Their efficacy in responding to presynaptic glutamate release depends on their kinetics, which are determined by AMPARs and their auxiliary subunit composition. /{beta}-Hydrolase domain-containing 6 (ABHD6) is an AMPAR auxiliary subunit that has been shown to negatively regulate the surface delivery of AMPARs and AMPAR-mediated currents. Overexpression of ABHD6 has been shown to decrease the rising slope and increase the decay {tau} of mEPSCs. However, whether ABHD6 is involved in regulating AMPAR kinetics remains unclear. Here, we found that ABHD6 itself had no effect on the gating kinetics of GluA1 and GluA2(Q) containing homomeric receptors. However, in the presence of the auxiliary subunit TARP {gamma}-2, ABHD6 accelerated the deactivation and desensitization of both GluA1 and GluA2(Q) containing homomeric receptors independent of their splicing isoforms (flip and flop) and the editing isoforms of GluA2 (R or G at position 764), except for the deactivation of GluA2(Q)i-G isoform. Besides, the recovery from desensitization of GluA1 with flip splicing isoform was slowed by the co-expression of ABHD6 in the presence of TARP {gamma}-2. Furthermore, ABHD6 accelerated the deactivation and desensitization of GluA1i/GluA2(R)i-G and GluA2(R)i-G/GluA3(R)i heteromeric receptors in the presence of TARP {gamma}-2. We also found that ABHD6-knockout neurons displayed slower deactivation and desensitization. Therefore, these results demonstrate that ABHD6 regulates AMPAR gating kinetics in a TARP {gamma}-2-dependent manner. SIGNIFICANCE STATEMENTThe efficacy of AMPARs in responding to presynaptic glutamate release depends on their kinetics, including deactivation, desensitization, and recovery from desensitization, which are determined by AMPARs and their auxiliary subunit composition. Using ultra-fast application of glutamate and outside-out patch recordings, we found that, in the presence of the auxiliary subunit TARP {gamma}-2, ABHD6 accelerated the deactivation and desensitization of GluA1i/GluA2(R)i-G and GluA2(R)i-G/GluA3(R)i heteromeric receptors and GluA1 and GluA2(Q) containing homomeric receptors independent of their splicing isoforms (flip and flop) and the editing isoforms of GluA2 (R or G at position 764), except for the deactivation of GluA2(Q)i-G isoform. In ABHD6 knockout neurons, we also observed slower deactivation and desensitization. These results demonstrated that ABHD6 regulates AMPAR gating kinetics in a TARP {gamma}-2-dependent manner.

neuroscience↗

bioRxiv2023

Genetic Dependence and Genetic Diseases

The human life depends on the function of proteins that are encoded by about twenty-thousand genes. The gene-disease associations in majority genes are unknown and the mechanisms underlying pathogenicity of genes/variants and common diseases remain unclear. We studied how human life depends on the genes, i.e., the genetic-dependence, which was classified as genetic-dependent nature (GDN, vital consequence of abolishing a gene), genetic-dependent quantity (GDQ, quantitative genetic function required for normal life), and genetic-dependent stage (GDS, temporal expression pattern). Each gene differs in genetic-dependent features, which determines the gene-disease association extensively. The GDN is associated with the pathogenic potential/feature of genes and the strength of pathogenicity. The GDQ-damage relation determines the pathogenicity of variants and subsequently the pathogenic genotype, phenotype spectrum, and inheritance of variants. The GDS is mainly associated with the onset age/evolution/outcome and the nature of genetic disorders (disease/susceptibility). The varied and quantitative genetic-dependent feature of genome explains common mild phenotype/susceptibility. The genetic-dependence discloses the mechanisms underlying pathogenicity of gene/variants and common diseases. One sentence summaryGenetic dependent feature differs in genes and determines pathogenicity of genes/variants and the clinical features of genetic diseases.

genetics↗

bioRxiv2023

BrrRCO, encoding a homeobox protein, is involved in leaf lobe development in Brassica rapa

Leaf shape is a vital economic and developmental trait in leafy vegetable Chinese cabbage (Brassica rapa L. subsp. pekinensis), which varies from smooth to deeply lobed, whereas the molecular basis remains unclear. Here, we detected an incompletely dominant major quantitative trait locus (QTL) qBrrLLA10 for the lobed-leaf trait in B. rapa, and identified BrrRCO, encoding a HD-Zip transcription factor, as the casual gene underlying lobed leaf formation in B. rapa. Genotyping analysis showed that abundant variations in promoter of BrrRCO is responsible for leaf lobe variation, and the expression levels and promoter activity were significantly affected by the promoter variations between two parents. BrrRCO was a nucleus-specific protein and possess highest expression level at the bases of each lobe. Ectopic overexpression of BrrRCO in Arabidopsis led to deeply lobed leaves have never been seen in wild type, and leaf lobe development was blocked when BrrRCO expression was down-regulated through virus-induced gene silencing assays. Taken together, our findings revealed that BrrRCO positively regulate leaf lobe formation in Chinese cabbage, and the cis-regulatory element modifications result in functional variation of BrrRCO, providing a novel insight into the leaf shape improvement in Chinese cabbage and other Brassica species. HighlightBrrRCO is essential for leaf lobe formation by repressing local growth of leaf margin cells in B. rapa, and cis-regulatory modifications cause the different allele effects.

plant biology↗

bioRxiv2023

Engineering cGAS-agonistic oligonucleotides as therapeutics and vaccine adjuvants for cancer immunotherapy

Current cancer immunotherapy (e.g., immune checkpoint blockade (ICB)) has only benefited a small subset of patients. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) activation holds the potential to improve cancer immunotherapy by eliciting type-I interferon (IFN-I) responses in cancer cells and myeloid cells. Yet, current approaches to this end, mostly by targeting STING, have marginal clinical therapeutic efficacy. Here, we report a cGAS-specific agonistic oligonucleotide, Svg3, as a novel approach to cGAS-STING activation for versatile cancer immunotherapy. Featured with a hairpin structure with consecutive guanosines flanking the stem, Svg3 binds to cGAS and enhances cGAS-Svg3 phase separation to form liquid-like droplets. This results in cGAS activation by Svg3 for robust and dose-dependent IFN-I responses, which outperforms several state-of-the-art STING agonists in murine and human immune cells, and human tumor tissues. Nanocarriers efficiently delivers Svg3 to tissues, cells, and cytosol where cGAS is located. Svg3 reduces tumor immunosuppression and potentiates ICB therapeutic efficacy of multiple syngeneic tumors, in wildtype but neither cGas-/- nor goldenticket Sting-/- mice. Further, as an immunostimulant adjuvant, Svg3 enhances the immunogenicity of peptide antigens to elicit potent T cell responses for robust ICB combination immunotherapy of tumors. Overall, cGAS-agonistic Svg3 is promising for versatile cancer combination immunotherapy.

bioengineering↗

bioRxiv2023

Insights from the Early Cretaceous: The promise of Lycoptera aDNA sequencing

Obtaining high-quality ancient DNA (aDNA) is a crucial process for advancements in molecular paleontology. However, current research is restricted to a limited range of fossil types due to DNA degradation and contamination from environmental DNA (eDNA). Utilizing the technology of nanoparticle affinity beads, we successfully extracted DNA from Lycoptera davidi fossils and obtained 1,258,901 DNA sequences. To ascertain their origins, we developed and implemented a novel protocol, the "mega screen method", designed to categorize aDNA fragments. This approach successfully identified 243 Actinopterygii species-specific fragments (SSFs), which subsequent analysis confirmed as original in situ DNA (oriDNA) sequences. Lycoptera davidi, a ray-finned fish from the Early Cretaceous Jehol Biota, dates back approximately 120 million years (Ma). Notably, the absence of deamination in certain oriDNA and paleoenvironmental DNA (paeDNA) fragments calls into question its reliability as a definitive marker for aDNA identification, a phenomenon potentially attributable to the protective effects of fossil diagenesis. Additionally, we identified a novel mechanism, "coding region sliding replication and recombination", responsible for generating new transposase. This mechanism, distinct from conventional processes such as horizontal gene transfer or vertical inheritance from parents, can facilitate a rapid genomic diversification. The transposase CDS insertion into the GAS2 gene of Anabarilius grahami offers molecular insights into the genetic interactions between ancestral carp and Lycoptera and the swift emergence of the expansion of early fish species starting from the early Cretaceous. These findings enhance our understanding of fossil DNA preservation, lay the groundwork for an ancient species DNA database, and introduce innovative tools for exploring evolutionary relationships across geological timescales.

paleontology↗

bioRxiv2023

GRAS1 non-coding RNA protects against DNA damage and cell death by binding NKAP

Non-coding RNA (ncRNA) gene products are involved in diverse biological processes including splicing, epigenetic regulation, gene expression, proliferation, and metabolism. The biological mechanisms by which ncRNAs contribute to cell survival remain poorly understood. We found that the Growth Regulator Antisense 1 (GRAS1) long non-coding RNA (lncRNA) transcript promotes growth in multiple human cell types by protecting against DNA damage. Knockdown of GRAS1 induced DNA damage and cell death, along with significant expression changes in DNA damage response, intrinsic apoptotic signaling, and cellular response to environmental stimulus genes. Extensive DNA damage occurred after GRAS1 knockdown, with numerous double strand breaks occurring in each cell. The number of cells undergoing apoptosis and with fragmented nuclei increased significantly after GRAS1 knockdown. We used RNA antisense purification and mass spectrometry (RAP-MS) to identify the NF-{kappa}B activating protein (NKAP) as a direct protein interaction partner of GRAS1 lncRNA. NKAP protein was degraded after GRAS1 knockdown, in a proteasome-dependent manner. Overexpression of GRAS1 or NKAP mitigated the DNA damage effects of GRAS1 knockdown. In summary, GRAS1 and NKAP directly interact to protect against DNA damage and cell death in multiple human cell lines.

molecular biology↗

bioRxiv2023

Cell division can accelerate the loss of a heteroplasmic mitochondrial DNA mutation in a mouse model of mitochondrial disease

Mitochondrial DNA (mtDNA) mutations accumulate in both mitotic and post-mitotic somatic tissues of normal individuals with age. They clonally expand within individual cells and cause mitochondrial dysfunction. In contrast, in patients with inherited disease-causing mtDNA mutations the mutation load decreases in mitotic tissues over time, whereas the mutations load in post-mitotic tissues remains relatively stable. The mechanisms underlying this decrease in mitotic tissues, and whether mitochondrial function is restored at the tissue level are unknown. Here, using a combination of homogenate tissue and single crypt/muscle fibre pyrosequencing we have shown a decrease in the mutation load of the germline heteroplasmic m.5024C>T mutation in multiple mitotic tissues of a mouse model of inherited mitochondrial disease (C5024T mice). We have then used in silico predictions to model the cellular dynamics of mtDNA mutation load in mitotic and post mitotic tissues. We demonstrate that: (1) the rate of m.5024C>T decrease correlates with the rate of tissue turnover; (2) the mutation load decrease is not associated with changes in overall cellular proliferation and apoptosis within the mitotic colonic epithelium; instead, it could be due to an upper limit of m.5024C>T load in stem cell populations; (3) the m.5024C>T mutation load is maintained in post-mitotic tissues over time with a consistent load amongst individual muscle fibres; (4) in silico modelling supports a scenario where genetic drift is accelerated in mitotic tissues by high levels of mtDNA replication coupled with mtDNA segregation at cell division. This study has advanced our understanding of the dynamics of mtDNA mutations and phenotype development in patients with mtDNA disease. Author SummaryHealthy individuals randomly accumulate pathogenic mtDNA mutations with age in dividing cells, causing mitochondrial dysfunction. Interestingly, patients with mitochondrial disease show a relative decrease in the loads of inherited mtDNA mutations in some dividing cells over time. The mechanisms underlying this decrease are unknown. Here we show a decrease in the load of the germline heteroplasmic m.5024C>T mutation in dividing cells and tissues of a mouse model of mitochondrial disease. In contrast, the mutation load in non-dividing cells and tissue remains stable. Our data are consistent with the hypothesis that a higher frequency of mtDNA replication in dividing cells, coupled with stem cells having an upper tolerance limit for m.5024C>T, causes an overall decrease in m.5024C>T load at the tissue level.

genetics↗