Biology subjects

Fong, N.

Publications and source records attributed to Fong, N..

12 recordsLinked to original sources

bioRxiv2026

AI discovery of sequence rules for RNA polymerase II pausing revises the pause-release model of gene activation

The promoter-proximal pause (PPP), at most metazoan genes, is believed to prime transcriptional activation by release of paused RNAPII into the gene. We tested the pause-release model by abolishing the PPP and monitoring gene activation. An LLM-guided AI discovered that upstream G's and a G T/C dinucleotide robustly discriminates pauses from non-pauses. Insertion of G-less sequences into NDRG1 and HSP90AA1 abolished the PPP in an orientation-dependent way, but did not impair activation by hypoxia or heat-shock. Hence pause-release is dispensable for these responses. The 5' peak of RNAPII detected by ChIP persisted even when pausing, detected by NET-seq was absent. Instead of pausing, these promoter-proximal polymerases may undergo abortive initiation. RNAPII C-terminal domain (CTD) deletion suppresses the PPP and shifts it upstream without affecting sequence-dependent pausing, consistent with promoter-tethering by this IDR. We propose that PPP formation is initiated by G-dependent arrest, and stabilized by multiple factors including CTD-dependent tethering.

molecular biology↗

bioRxiv2026

Widespread exon definition is promoted by the U1 snRNP 70K subunit and requires sites of contact with RNA Polymerase II

Much pre-mRNA splicing initiates co-transcriptionally, but its mechanism is unclear. We investigated this process by degron depletion of the U1-70K subunit of U1 snRNP that contacts transcribing RNA polymerase II (RNAPII). U1-70K deficiency caused extensive exon skipping consistent with widespread exon definition. Surprisingly, exons with the strongest 5' splice sites (SSs) are skipped. Furthermore, strengthening of non-canonical 5' splice sites with a complementary mutant U1 snRNA or a splice modifying drug induced exon skipping in response to U1-70K depletion. We suggest that U1-70K plays a previously unappreciated role in destabilizing the U1-5'SS interaction to facilitate the transition to the U6-5'SS interaction required for productive splicing following exon definition. Consistent with this idea, knock- down of Prp28/DDX23 that exchanges U1 for U6 at the 5'SS also promotes exon skipping mimicking U1-70K depletion. Replacing WT U1-70K in degron-containing cells with a mutant in the RNAPII interface caused widespread exon skipping. We propose that frequent exon definition occurs at the transcription elongation complex and is enabled by U1-70K contact with transcribing RNAPII.

molecular biology↗

bioRxiv2026

Dynamic interaction between tentacle-tethered microdomains in Integrator and NELF regulates the promoter-proximal pause checkpoint

Negative Elongation Factor (NELF) and Integrator are general regulators of transcriptional pausing and premature termination at the beginning of protein-coding genes. While NELF is a key pausing factor, Integrator triggers premature termination of dysfunctional RNAPII complexes. Here, we identify a direct interaction between the plant homeodomain (PHD) finger of Integrator subunit 12 (INTS12) and a previously unrecognized C-terminal domain (CTD) in the flexible tentacle of NELF-A. Mutation of either domain disrupts the interaction, and although RNAPII-associated pausing complexes still form, they are dysfunctional. Importantly, while deletion of the INTS12 PHD finger functionally mimics Integrator loss, a single point mutation in NELF-A CTD instead mimics loss of NELF, suggesting the presence of competitive interactions. Together, our findings uncover a novel mode of PHD finger binding and outline how an interaction between Integrator and NELF regulates transcriptional pausing and premature termination.

cell biology↗

bioRxiv2026

A new phased assembly of the Antarctic spiny plunderfish provides novel insights into the evolution of the notothenioid radiation.

BackgroundNotothenioids are a well characterised species flock endemic to the Antarctic and an important model group for the study of genome adaptation to extreme cold. We used a new reference assembly and clade-wide comparative genomic analysis to investigate cryonotothenioid evolution and the appearance of novel functionalities linked to cold adaptation. ResultsA new phased assembly of a model notothenioid, Harpagifer antarcticus, demonstrated low levels of haplotypic variability across the genome. Nevertheless, numerous insertions from multiple LINE-L2 clades were found, suggesting ongoing transposition with potential contribution to speciation. Contrary to expectations the afgp locus was highly similar between haplotypes, except for large length allelic variants of afgp genes. Analysis suggests a model for the afgp locus expansion in H. antarcticus through segmental tandem duplications involving two pairs of afgp genes at time. Syntenic reconstruction of genomes from across the clade demonstrates conserved macrosyntenic relationships and group specific chromosomal fusions of notothenioids. Quantification of genome gain and transposition rates during cryonotothenioid diversification showed a first ancestral slow genome expansion concurrent with historic temperature drops. This was followed by lineage-specific massive peaks of genomic gain and transposition activity. Finally, we identified a set of genes that underwent ancestral diversifying selection and acquired novel conserved non-coding elements during the cryonotothenioid emergence. These were related to antioxidants and proteostasis, which may have facilitated the notothenioid Antarctic radiation. ConclusionDiversifying selection and genomic gain linked to transposon activity are primary contributors to lineage-specific evolutionary dynamics through the clade which facilitated adaptation to life in the cold.

genomics↗

bioRxiv2026

PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

Metastasis remains the leading cause of cancer mortality, yet effective therapies are limited. While therapeutic responses are influenced by organ-specific immune microenvironments, strategies to pharmacologically modulate these niches remain poorly defined. Here, using the clinical-stage inhibitor ABBV-CLS-484 (AC484) as a chemical probe, we demonstrate that systemic PTPN1/2 inhibition remodels the pulmonary myeloid landscape, specifically activating alveolar macrophages (AMs) toward a tumoricidal state. Integrated single-cell/spatial transcriptomics and functional assays reveal that AC484 promotes AM accumulation within metastatic lesions, elevates their IFN{gamma} production and responsiveness, and enhances their tumor-killing activity. Depletion of AMs diminishes the anti-metastatic efficacy of AC484. Mechanistically, inhibiting PTPN1/2 by AC484 amplifies IFN{gamma}-STAT1 signaling in AMs, while disrupting this pathway impairs their tumor control capability. These findings delineate a distinct innate immune axis where PTPN1/2 acts as a molecular "brake" on AM activation, suggesting that pharmacologically unleashing tissue-resident macrophages offers a therapeutic strategy to overcome metastatic progression, particularly in microenvironments where adaptive immunity is insufficient. SignificanceAMs are potent anti-metastatic immune effectors functionally constrained by PTPN1/2. Pharmacologically inhibiting PTPN1/2 amplifies their IFN{gamma}-STAT1 signaling and tumoricidal activity, establishing a strategy to reactivate tissue-resident immunity against metastasis.

immunology↗

bioRxiv2025

Heterochronic myeloid cell replacement reveals the local brain environment as key driver of microglia aging

Aging, the key risk factor for cognitive decline, impacts the brain in a region-specific manner, with microglia among the most affected cell types. However, it remains unclear whether this is intrinsically mediated or driven by age-related changes in neighboring cells. Here, we describe a scalable, genetically modifiable system for in vivo heterochronic myeloid cell replacement. We find reconstituted myeloid cells adopt region-specific transcriptional, morphological and tiling profiles characteristic of resident microglia. Young donor cells in aged brains rapidly acquired aging phenotypes, particularly in the cerebellum, while old cells in young brains adopted youthful profiles. We identified STAT1-mediated signaling as one axis controlling microglia aging, as STAT1-loss prevented aging trajectories in reconstituted cells. Spatial transcriptomics combined with cell ablation models identified rare natural killer cells as necessary drivers of interferon signaling in aged microglia. These findings establish the local environment, rather than cell-autonomous programming, as a primary driver of microglia aging phenotypes.

neuroscience↗

bioRxiv2025

CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing

Pre-mRNA splicing is kinetically coupled to transcription as shown by the widespread effects of transcription speed on alternative splicing outcomes. The molecular basis for such kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate dependent alternative folding pathways of the nascent pre-mRNA. To search for RNA structures in nascent pre-mRNA, we modified SPLASH (1) (Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids) for use with Chromatin Associated RNA. We applied this new method called CAR-SPLASH to cells expressing WT and slow mutant RNA polymerase II and identified > 3000 intramolecular RNA structures of which > 400 are proximal to splice sites. Antisense oligonucleotide (ASO) disruption of several such structures that sequester splice sites has a major impact on alternative splicing outcomes, even though the ASOs do not directly disrupt splice sites. ASO disruption of novel regulatory elements that we designate "RNA kinetic switches", modified alternative splicing of NISCH Exon 18, GAK Exon 7 and MEGF8 Exon 14 in a way that depends on the rate of transcription elongation. We propose that these switches mediate kinetic coupling via effects of transcription speed on folding of nascent RNA structures that modulate alternative splicing, and that many nascent RNA structures can thereby serve as new targets for splice modifying ASOs. Significance StatementAlternative splicing (AS) of mRNAs is a major regulator of gene expression that is frequently disrupted is disease. AS is affected by the speed of RNA polymerase II that synthesizes mRNA precursors, but how such kinetic coupling works is not well understood. We developed a method, CAR-SPLASH, to capture RNA structures in nascent RNA chains by cross-linking with psoralen. CAR-SPLASH identified novel structural regulatory elements we call "RNA kinetic switches" that control AS in a way that depends on the speed of transcription. RNA kinetic switches are new targets for splice modifying antisense oligonucleotides that could have therapeutic value.

molecular biology↗

bioRxiv2024

PP1 PNUTS binds the restrictor and dephosphorylates RNA pol II CTD Ser5 to stimulate transcription termination

The restrictor, ZC3H4/WDR82, is the major termination factor for antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 co-purifies with PP1 phosphatase and PP1 phosphatase nuclear targeting subunit, PNUTS, which binds directly to the WDR82 subunit of restrictor. AlphaFold predicts a quaternary complex, PPWZ, in which PP1-associated PNUTS and ZC3H4 both contact WDR82. To investigate the role of protein dephosphorylation in PPWZ activity, we expressed a substrate trap comprising inactive PP1H66K linked to the PNUTS C-terminus. PP1H66K-PNUTS binds pol II large subunit and nuclear exosome components. PP1H66K-PNUTS, but not PP1WT-PNUTS, functions as a dominant-negative inhibitor of antisense termination and CTD Ser5 dephosphorylation. Both these activities require the PNUTS WDR82 binding domain that interacts with restrictor. We show that CTD Ser5 hyperphosphorylation is associated with higher processivity and reduced pausing that would counteract termination, and propose that Ser5 dephosphorylation by PPWZ is coupled to termination. In summary, we identify the PP1 phosphatase activity of the PPWZ complex as essential for terminator function and propose that this heterotetramer is the physiologically relevant form of restrictor.

molecular biology↗

bioRxiv2024

Design principles for accurate folding of DNA origami

We describe design principles for accurate folding of three-dimensional DNA origami. To evaluate design rules, we reduced the problem of DNA strand routing to the known problem of shortest-path finding in a weighted graph. To score candidate DNA strand routes we used a thermodynamic model that accounts for enthalpic and entropic contributions of initial binding, hybridization, and DNA loop closure. We encoded and analyzed new and previously reported design heuristics. Using design principles emerging from this analysis, we redesigned and fabricated multiple shapes and compared their folding accuracy using electrophoretic mobility analysis and electron microscopy imaging. We demonstrate accurate folding can be achieved by optimizing staple routes using our model, and provide a computational framework for applying our methodology to any design.

biophysics↗

bioRxiv2022

Nucleotide level linkage of transcriptional elongation and polyadenylation

Alternative polyadenylation yields many mRNA isoforms whose 3 termini occur disproportionately in clusters within 3 UTRs. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. In yeast, upstream and downstream shifts within isoform clusters occur steadily at the nucleotide level. In contrast, changes from one isoform to the next are much smaller between clusters, even when the distances between them are relatively large. GC content in a region 13-30 nt downstream from isoform clusters is linked to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at the nucleotide level within clusters, but not between them. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

molecular biology↗

bioRxiv2020

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival

The sequence content of the 3' UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. To combat this problem, we developed LABRAT, an APA quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between APA and expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated.

genomics↗

bioRxiv2019

Solo: doublet identification via semi-supervised deep learning

AO_SCPLOWBSTRACTC_SCPLOWSingle cell RNA-seq (scRNA-seq) measurements of gene expression enable an unprecedented high-resolution view into cellular state. However, current methods often result in two or more cells that share the same cell-identifying barcode; these "doublets" violate the fundamental premise of single cell technology and can lead to incorrect inferences. Here, we describe Solo, a semi-supervised deep learning approach that identifies doublets with greater accuracy than existing methods. Solo can be applied in combination with experimental doublet detection methods to further purify scRNA-seq data to true single cells beyond any previous approach.

bioinformatics↗