Biology subjects

Jia, W.

Publications and source records attributed to Jia, W..

At least 19 recordsLinked to original sources

bioRxiv2026

3D genome remodelling underlies PU.1-dependent granulocyte maturation

PU.1 is an ETS-domain transcription factor that has critical roles in many aspects of hematopoiesis and immune cell fate and function. In addition, aberrant PU.1 expression has been implicated in the development of acute myeloid leukemia (AML). Loss of PU.1 during adult murine hematopoiesis results in the expansion of immature granulocytes suggesting that PU.1 plays an important role in granulocyte maturation. To understand the molecular underpinnings of this process, we combined gene expression, transcription factor binding and 3D genome analyses with conditional deletion of PU.1 in vivo. We find that in contrast to normal granulocytes, PU.1-deficient cells possessed a transcriptome of immature granulocytes, in line with their cellular phenotype. Furthermore PU.1-deficient granulocytes display altered 3D genome architecture with a significant loss of interactivity in regions bound by PU.1 in control cells. Overall, this study implicates PU.1 as a key regulator of granulocyte maturation and lineage commitment through control of transcriptional programs and 3D chromatin architecture.

immunology↗

bioRxiv2026

A human intracranial map of consciousness returning from anesthesia

How consciousness arises from the brain remains a central yet unsolved question. Although substantial effort has been devoted to characterizing the neural correlates of conscious perception during wakefulness, how consciousness re-emerges from unconscious states remains poorly understood. Here, we took advantage of the rare opportunity to record human intracranial local field potentials from propofol-induced general anesthesia through the transition to behavioral engagement and subsequent full wakefulness, capturing both the stable states of unconsciousness and consciousness and the reconstituting neural dynamics that bridge them. Anesthesia cannot be defined by any single electrophysiological signature; rather, it is an organized low-frequency regime characterized by a constellation of coordinated neural phenomena, including aperiodic slow waves, alpha/beta periodicity, global alpha synchronization, and slow-wave-alpha/beta phase-amplitude coupling. Following anesthetic cessation, this regime progressively dissolved as neural excitability and dynamical complexity climbed. The appearance of conscious behavior coincided with a fast and dramatic transformation in high-gamma activity, from stochastic and unpredictable bursts to structured responses that were distributed, task-selective, and event-locked. These findings suggest that the recovery of consciousness is a multiscale reorganization in which distinct neural underpinnings rise and fall. Together, this work charts an electrophysiological map of how conscious cognition is extinguished, reconfigured, and restored in the human brain.

neuroscience↗

bioRxiv2026

Decoding heterogeneous aging clocks and disease risk stratification using a metabolomic foundation model

Metabolomic aging clocks estimate biological age by modeling metabolite concentrations, thereby capturing aging signals from healthspan and adverse outcomes. However, existing clocks generally assume homogeneous aging trajectories and yield only a single age acceleration metric, limiting their capacity to capture inter-individual metabolic heterogeneity and characterize nuanced individual-level representations. To address these limitations, we proposed MetAgeFormer, a transformer-based metabolomic model pre-trained on nuclear magnetic resonance (NMR) metabolomic profiles from over 430,000 participants in UK Biobank via self-supervised learning. This large-scale pre-training enables MetAgeFormer to learn a metabolomic representation space that captures the complex, nonlinear structure of systemic metabolism as reflected in NMR data. Building on MetAgeFormer, we developed a mortality-informed metabolomic aging clock by fine-tuning an attached survival module, deriving age acceleration that demonstrates significant associations with multiple age-related diseases and factors. We further validated zero-shot transfer in the independent Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. More importantly, we utilized embeddings generated by MetAgeFormer to identify 13 distinct metabolic subtypes and consolidated them into four meta-subtypes with markedly divergent susceptibility profiles for major age-related diseases, particularly type 2 diabetes and neurodegenerative disorders. This finding empirically demonstrated substantial metabolic heterogeneity across populations, persisting even at comparable levels of age acceleration. To enhance clinical applicability, we further employed contrastive learning to distill a lightweight model that approximates the learned metabolomic representation space using only 14 routine clinical blood test measurements as inputs. Both hold-out testing within UK Biobank and external validation in the China Health and Retirement Longitudinal Study replicated similar disease onset patterns across the identified subtypes, underscoring the robust generalizability of MetAgeFormer and supporting its translational potential as a scalable framework for metabolomic aging assessment and early disease risk stratification.

bioinformatics↗

bioRxiv2025

MPKaDB: A pKa Database for Exploring pH Dependence in Membrane Proteins

The biological activities of many membrane proteins are pH-regulated, yet mapping their pH dependence experimentally is slow and expensive. In this work, we present MPKaDB (http://computbiophys.com/DeepKa/mpkadb), a comprehensive pKa database for membrane proteins that instantly decode the protonation states of ionizable residues under a specified pH. Leveraging MPKaDB, we performed pH-coupled electrostatic characterization of trans-membrane proteins. To facilitate use, a user-friendly search engine was developed to retrieve a protein of interest and returns its pKa values, isoelectric points of both cytoplasmic and extra-cytoplasmic faces, and an automated screening of active-site residues. In the end, two case studies were proposed to demonstrate how pKas from MPKaDB could be applied to explore the pH-dependent relationship between membrane protein structure and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/688495v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@f90726org.highwire.dtl.DTLVardef@1448ac2org.highwire.dtl.DTLVardef@f1e4f4org.highwire.dtl.DTLVardef@ee57dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Disruption of ADAMTSL4 Causes Ectopia Pupillae in Zebrafish via COL8A1-Driven Cell Migration

Congenital ectopia lentis (EL) poses a significant threat to visual outcomes in children. Although emerging causative genes have been identified, their biological functions remain poorly understood. In this study, we investigated the contribution of ADAMTSL4 variants to congenital EL in a cohort of 702 pedigrees. Pathogenic variants in ADAMTSL4 were identified in 23 probands (3.28%), comprising 29 distinct variants, with 14 being novel, making ADAMTSL4 the second most frequently mutated gene in the cohort. All affected individuals exhibited EL, and 26.09% also presented with ectopia pupillae (EP), also referred to as ectopia lentis et pupillae. To explore the functional consequences of ADAMTSL4 deficiency, we generated a CRISPR/Cas9-mediated adamtsl4-knockout zebrafish model, that faithfully recapitulated cardinal human disease features with an incidence comparable to that observed in affected patients. Histological and ultrastructural analysis revealed disrupted zonular fiber anchorage at the lens capsule, even in mutated eyes without overt EL or EP. Transgenic overexpression of adamtsl4 successfully reversed the ocular phenotypes, confirming the genes essential role in ocular development. Single-cell RNA-sequencing and fluorescence in situ hybridization demonstrated enriched ADAMTSL4/adamtsl4 expression in the equatorial lens epithelium, retinal pigment epithelium (RPE), iris anterior pigmented epithelium, and choroid fibroblast. Functional assays using zebrafish and human RPEs revealed that ADAMTSL4 deficiency compromised cell adhesion and promoted cell migration. Transcriptomic profiling revealed significant enrichment of extracellular matrix organization and cell adhesion pathways, with cross-species validation identifying consistent upregulation of COL8A1/col8a1b. Notably, COL8A1 knockdown in ADAMTSL4-deficient RPEs partially reversed the aberrant migratory phenotype, suggesting a functional interaction. Together, these findings establish ADAMTSL4 as a major causative gene in ectopia lentis et pupillae, highlight its role in orchestrating ocular integrity via regulation of extracellular matrix and cell behavior.

genetics↗

bioRxiv2025

Activation of the noncanonical inflammasome-GSDMD pathway triggers pyroptosis in bone marrow and promotes periosteal bone formation

Evidence indicating that inflammation is commonly associated with ectopic osteogenesis in certain autoimmune and infectious conditions challenges the dogma that inflammatory responses always suppress bone formation. In this study, we find that systemic administration of lipopolysaccharide (LPS) to mice causes not only inflammation in bone marrow, as expected, but also stimulates periosteal bone formation. This response can be reproduced in vitro as bone marrow supernatants from LPS-treated mice induce robust osteogenesis of skeletal stem cells (SSCs) compared to supernatants from PBS-treated counterparts. Periosteal bone accrual is partly dependent on periosteal leptin receptor-positive (LepR)+ SSCs but not bone marrow LepR+ or adiponectin (Adq)+ SSCs and correlates with pyroptosis within bone marrow. Consistent with the dependence of periosteal osteogenesis on pyroptosis, this response is slightly attenuated in Nlrp3-/- or caspase-1-/-mice but significantly inhibited in caspase-11-/-, caspase-1-/-;caspase-11-/-, or Gsdmd-/-mice. Our study reveals a novel role for pyroptosis in which lysed cells release intracellular contents that stimulate osteoprogenitors and promote osteogenic differentiation within the periosteal compartment.

cell biology↗

bioRxiv2025

BCKDK, a novel hypoxia-responsive kinase that exacerbates cerebral ischemia injury

Alterations of circulating amino acid profile have been observed in patients with ischemic stroke. However, whether ischemia disrupts amino acid metabolism in the brain tissue and subsequently potentiates cellular stress and cerebral injury have never been explored. Employing a metabolomics approach combined with metabolic flux analysis, impaired catabolism and significant enrichment of branched-chain amino acids (BCAAs) were identified in mouse primary neuron cells upon oxygen-glucose deprivation. Consistently, BCAA catabolism was also damaged in the brain of mouse with acute ischemic stroke, accompanied with suppressed activity of branched-chain alpha-keto acid dehydrogenase (BCKDH) and upregulation of BCKDH kinase (BCKDK). Furthermore, restoration of BCAA catabolism by suppressing BCKDK via pharmacological inhibitor or silencing RNA dramatically alleviated cerebral ischemia injury in mice. Mechanistically, ischemia induces the expression of BCKDK via hypoxia-inducible factor 1-mediated transcriptional activation and inhibits BCAA conversion into substrates for tricarboxylic acid cycle, contributing to potentiated energy deficiency, glutamate excitotoxicity, and neuronal injury. Collectively, this study identified BCKDK as a novel hypoxia-responsive factor and a promising therapeutic target for cerebral ischemia injury.

molecular biology↗

bioRxiv2025

Dynamic connectivities of plant metacommunities at a millennial time-scale: the Beringia testbed

Ecological connectivity shapes ecosystem responses to climate change and is thought to underpin stability, yet its millennial-scale dynamics remain poorly resolved. We asked how spatial and temporal connectivity of plant metacommunities changed over the last 40 ka and which processes drove it. We analysed and compiled plant sedimentary DNA from 20 lake cores across Beringia (Siberia, Alaska) to investigate community dynamics and, for a high-resolution subset, applied beta- and zeta-diversity to track connectivity. Vegetation changed coherently across the glacial-Holocene transition, with trait shifts mirroring functional composition. Connectivity peaked during the late MIS3 and Last Glacial Maximum--likely aided by the Bering Land Bridge, mass effects and facilitation--collapsed during the Deglacial with rapid turnover, and rebounded in the Holocene as shrub and boreal communities expanded. Temporal zeta within sites exceeded spatial zeta, indicating strong local persistence and resilience. Tundra sites uninvaded by forest maintained continuous species pools. Overall, these patterns underscore the value of a metacommunity perspective for assessing millennial-scale connectivity changes.

ecology↗

bioRxiv2025

Cooperativity and Communication between the Active Sites of the Dimeric SARS-CoV-2 Main Protease

The coronaviral main protease (Mpro) is essential for the replication of the virus, and has been the subject of various biochemical, structural and enzymatic studies, as well as a drug target against SARS-CoV-2 infections. SARS-CoV-2 Mpro is known to be active as a dimer, with the N terminus of one protomer completing a key active site pocket of the other protomer. Despite apparent cooperativity in catalytic activity, how the two distal active sites in the dimer communicate and might be modulating binding and/or catalysis at the other remain to be clarified. Here, we have investigated the interplay between cooperativity, dimerization, and substrate cleavage in SARS-CoV-2 Mpro through a combination of enzymatic assays, crystal structures, and protein characterization. To disentangle the contribution of each active site to the observed enzymatic activity, we developed a cleavage assay involving heterodimers of active and inactive (C145A or inhibitor-bound) monomers. Strikingly, we found that heterodimerization increased cleavage efficiency per active monomer. Additionally, we mapped a network of critical residues bridging the two active sites and probed this network through engineered mutations. By dissecting the cooperativity and communication between the active sites, we provide new insights into the Mpro reaction cycle and functional significance of its dimeric architecture.

biochemistry↗

bioRxiv2025

Immune cells regulate circulating adipocyte extracellular vesicle levels in response to metabolic shifts

Extracellular vesicles (EVs) are now recognized as potent mediators of intercellular and inter-organ signaling and implicated in the pathogenesis of obesity and its associated comorbidities such as diabetes, cancer, cardiovascular disease, and neurodegeneration. Despite a surge of new functional information about EVs, we still lack a basic understanding of how endogenous EV levels are controlled to regulate inter-organ signaling. New flow cytometry technology has allowed us to study the regulation of circulating, endogenous EVs from metabolically relevant cell types like adipocytes. From this, we provide evidence for a paradigm of EV regulation where tissue resident immune cells, predominantly macrophages, clear EVs released by local tissue cells or EVs entering the tissue from circulation, an activity that determines circulating EV levels. In obesity, EV uptake by adipose tissue immune cells is reduced, concomitant with increased circulating adipocyte-specific EVs (adipoEVs) and reduced EV clearance rates. AdipoEVs are significantly elevated in mouse circulation from one day to 20 weeks of high-fat feeding. In humans we found that adipocyte EV levels negatively correlate with whole-body and liver insulin sensitivity and are not associated with adipose mass. This work suggests that tissue resident immune cells act as a gatekeeper for tissue EV entry into circulation and are thereby a major regulator of inter-organ EV signaling.

physiology↗

bioRxiv2025

Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

Tumor-infiltrating neutrophils (TINs) are highly heterogeneous and mostly immunosuppressive in the tumor immune microenvironment (TIME). Current biomarkers of TINs and treatment strategies targeting TINs have not yielded optimal responses in patients across cancer types. Here, we separated human and mouse neutrophils into three developmental stages, including promyelocyte (PM), myelocyte & metamyelocyte (MC & MM), and banded & segmented (BD & SC) neutrophils. Based on this separation, we observed the predominance of human but not mouse MC & MM-stage neutrophils in bone marrow (BM), which exhibit potent immunosuppressive and tumor-promoting properties. MCs & MMs also occupy the majority of TINs among patients with 17 cancer types. Moreover, through the creation of a NCG-Gfi1-/- human immune system (HIS) mouse model, which supports efficient reconstitution of human TIN, we found a significant increase of BM MCs & MMs in tumor-bearing mice. By comparing the scRNA-seq analysis results of human neutrophils from both BM and tumors, we found CD63 and Galectin-3 distinguish MC & MM from neutrophil populations in cancer patients. Furthermore, we proposed a strategy with Flt3L to specifically induce the trans-differentiation of MCs & MMs into monocytic cells, and trigger tumor control in NCG-Gfi1-/- HIS mice. Thus, our findings establish an essential role of human MC & MM-stage neutrophils in promoting cancer progression, and suggest their potential as targets for developing potential biomarkers and immunotherapies for cancer.

immunology↗

bioRxiv2025

Woody plant encroachment enhances negative interactions in Arctic plant communities over the last 24,000 years

Woody taxa encroachment in the Arctic has been widely observed. However, it remains uncertain how plant interactions are affected by such encroachment due to the lack of long-term observational data. Here, we reconstruct plant composition and functional trait turnover during post-glacial woody encroachment using sedimentary ancient DNA from nine lakes in the Siberia-Alaska region. Environmentally constrained null models are applied to partition plant interactions from the pure environment driven plant co-occurrence signal. Our results show that plant interactions shifted from predominantly positive interactions (e.g. nurse-plant facilitation) during the glacial period to negative interactions (e.g. competition) during the Holocene. This shift coincided with a community transition from herbaceous to woody taxa, leading to an increase in average plant height and root length, as evidenced by leveraging plant trait information. We suggest that climate (an external factor) and plant interactions (an internal process) jointly supported rapid and widespread woody taxa expansion at the end of the last glacial, which may provide an analogy with contemporary "arctic greening". In turn, woody encroachment is likely to constrain the geographical ranges of native species, increasing the risk of local native taxa loss, while enhancing beta diversity.

ecology↗

bioRxiv2025

Exploring Population Differences in the Human Gut Microbiome: from Microbial Abundance to Single Nucleotide Polymorphisms

The human gut microbiota exhibits significant diversity across populations, influenced by factors such as geography, diet, and lifestyle, particularly between the Han Chinese and non-Chinese populations. While previous studies have predominantly focused on the taxonomic abundance of the gut microbiome, the impact of single nucleotide polymorphisms (SNPs) in driving population-specific differences remains largely underexplored. In this study, we systematically investigated gut microbial differences between the Han Chinese and non-Chinese populations using the Human Gut Microbiome Reference Genome Catalog (HGMRGC). HGMRGC includes 271,480 non-redundant genomes from 5,785 prokaryotic species, which was generated by metagenomic sequencing data from 9,320 publicly available samples across 22 countries and 3,584 newly sequenced samples from Han Chinese individuals across 29 provinces and regions in China. We observed geography was the primary driver of microbial variation of abundance and SNPs. We identified 625 novel population-specific genome clusters from HGMRGC with functional differences in carbohydrate utilization and 126 species exhibiting distinct prevalence related to vitamin biosynthesis, antibiotic resistance, and carbohydrate metabolism. Beta diversity analysis highlighted significant inter-population differences in both microbial abundance and SNPs, while alpha diversity analysis revealed that non-Chinese populations exhibited higher diversity in microbial abundance, and Han Chinese populations displayed greater diversity in SNPs. These results provide valuable insights into population-specific microbial diversity, laying the groundwork for future research on its functional and health implications.

microbiology↗

bioRxiv2025

Therapeutic Potential of 20E in Pulmonary Arterial Hypertension: Involvement of Mas Receptor and PI3K/Akt Pathway

BACKGROUND20-Hydroxyecdysone (20E), a natural polyhydroxylated steroid found in various edible plants, exhibits diverse pharmacological effects. This study aims to investigate the preventive and therapeutic effects of 20E on pulmonary arterial hypertension (PAH) and elucidate its underlying molecular mechanisms. METHODSA monocrotaline (MCT)-induced PAH rat model was utilized to evaluate the efficacy of 20E. The Mas receptor antagonist A779 and agonist AVE0991 were used to investigate the role of Mas in PAH progression and 20E-mediated prevention. Molecular docking and pull-down assays were conducted to confirm the interaction between 20E and the Mas receptor. In vitro, the effects of 20E on Ang II-induced proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) were assessed. The PI3K/Akt signaling pathway was analyzed by Western blot. RESULTS20E prevented PAH at 30 mg/kg and 90 mg/kg, while 90 mg/kg rescued pre-existing PAH. The protective effects of 20E were attenuated by A779. 20E upregulated Mas receptor expression and directly bound to it. In vitro, 20E inhibited Ang II-induced HPASMC proliferation and migration. It also downregulated p-PI3K, p-Akt, and p-mTOR while restoring P27 and P21 expression. Furthermore, knockdown of the Mas in HPASMCs abolished the effects of 20E on these processes. CONCLUSIONS20E inhibits PASMC proliferation and migration by activating the Mas receptor and modulating the downstream PI3K/Akt signaling pathway, thereby effectively preventing and rescuing PAH. It may be a promising pharmacological candidate for PAH treatment.

molecular biology↗

bioRxiv2025

Molecular Mechanisms of Drug Resistance and Compensation in SARS-CoV-2 Main Protease: The Interplay Between E166 and L50

The SARS-CoV-2 main protease (Mpro) is essential for viral replication, and a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the Mpro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged in in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to 2- fold. This loss of catalytic efficiency was compensated for by the addition of L50F in the double- mutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wild- type demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 side- chain interactions to minimize susceptibility to resistance.

biochemistry↗

bioRxiv2024

Leveraging Drosophila Models to Explore AI-generated Synthetic Peptide's Potential in Boosting Honeybee Health and Resilience

The integration of artificial intelligence (AI) and machine learning (ML) in peptide design has revolutionized the development of antimicrobial peptides (AMPs), which are essential components of innate immunity. In this study, we identified a novel synthetic peptide, PAN4 (GAYTFKIRRK), through genetic screening of AI-generated candidates in Drosophila melanogaster. PAN4 demonstrated robust antimicrobial activity, stress tolerance, and antitumor effects, significantly enhancing survival rates following bacterial infections and improving locomotor behaviors without adversely affecting lifespan. Furthermore, PAN4 expression in intestinal stem cells completely suppressed RasV12-induced tumor progression, indicating its potential role in cancer prevention. The peptide also mitigated gut barrier dysfunction associated with sleep deprivation and reduced inflammation in a dextran sulfate sodium (DSS)-induced colitis model. Mechanistically, PAN4s antimicrobial activity was linked to its interaction with specific peptidoglycan recognition proteins (PGRPs), particularly PGRP-SC1a, while the Tak1-mediated immune signaling pathway was found to be non-essential for its efficacy. PAN4 showed promising effects on honeybee health, enhancing survival rates under bacterial stress. Furthermore, PAN4 expression demonstrated significant anti-tumor activity in the Drosophila gut tumor model. Our findings suggest that PAN4 serves as a versatile agent with significant implications for enhancing immune responses and combating diseases in honeybee populations, paving the way for future applications in agriculture and medicine.

genetics↗

bioRxiv2024

Deep Ensemble Model for Quantitative Optical Property and Chromophore Concentration Images of Biological Tissues

The ability to quantify widefield tissue optical properties (OPs, i.e., absorption and scattering) has major implications on the characterization of various physiological and disease processes. However, conventional image processing methods for tissue optical properties are either limited to qualitative analysis, or have tradeoffs in speed and accuracy. The key to quantification of optical properties is the extraction of amplitude maps from reflectance images under sinusoidal illumination of different spatial frequencies. Conventional three-phase demodulation (TPD) method has been demonstrated for the mapping of OPs, but it requires as many as 14 measurement images for accurate OP extraction, which leads to limited throughput and hinders practical translation. Although single-phase demodulation (SPD) method has been proposed to map OPs with a single measurement image, it is typically subject to image artifacts and decreased measurement accuracy. To tackle those challenges, here we develop a deep ensemble model (DEM) that can map tissue optical properties with high accuracy in a single snapshot, increasing the measurement speed by 14x compared to conventional TPD method. The proposed method was validated with measurements on an array of optical phantoms, ex vivo tissues, and in vivo tissues. The errors for OP extraction were 0.83 {+/-} 5.0% for absorption and 0.40 {+/-} 1.9% for reduced scattering, dramatically lower than that of the state-of-the-art SPD method (2.5 {+/-} 15% for absorption and -1.2 {+/-} 11% for reduced scattering). It was further demonstrated that while trained with data from a single wavelength, the DEM can be directly applied to other wavelengths and effectively obtain optical property and chromophore concentration images of biological tissues. Together, these results highlight the potential of DEM to enable new capabilities for quantitative monitoring of tissue physiological and disease processes.

biophysics↗

bioRxiv2024

Alternative splicing modulates chromatin interactome and phase separation of the RIF1 C-terminal domain

How RIF1 (RAP1 interacting factor) fulfills its diverse roles in DNA double-strand break (DSB) repair, DNA replication, and nuclear organization remains elusive. Here we show that alternative splicing (AS) of a cassette exon (Ex32) encoding a Ser/Lys-rich (S/K) cassette in the RIF1 C-terminal domain (CTD) gives rise to RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with different functional characteristics. We demonstrate that RIF1-Ex32 splice-in is mediated by an exonic splicing enhancer that is recognized by the splicing factor SRSF1 and antagonized by splicing inhibitors SRSF3 and SRSF7. Exposure to DNA damage inhibited Ex32 splice-in, potentiated the association of SRSF3 and SRSF7 with RIF1 pre-mRNA, and caused an increase in RIF1-S protein expression, which was also observed across a diverse set of primary cancers. Isoform-specific proteomic analyses revealed RIF1-L preferentially associated with mediator of DNA damage checkpoint 1 (MDC1) and sustained MDC1 focus formation to a greater extent than RIF1-S. We further show that the S/K cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by CDK1-dependent phosphorylation of the RIF1 CTD in response to G2 DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 AS contributes to RIF1 functional diversification in genome protection.

molecular biology↗