Biology subjects

Sharp, P. A.

Publications and source records attributed to Sharp, P. A..

12 recordsLinked to original sources

bioRxiv2026

Neural RNA splicing models rely on reading frame information unavailable to the splicing machinery

Deep neural networks can accurately predict RNA splice sites from genomic sequence, but whether they do so by simulating the natural splicing process is unclear. We evaluate this question by examining mutations that should not have a strong effect on splicing, but are correlated with it in the human genome. We focus specifically on mutations that affect frame alignment because this is known to be relevant to machinery that operates spatially and temporally separately from the splicing machinery. We find that predictions made by commonly-used neural tools are impacted far more by deletions or mutations that alter translational reading frame, producing in-frame stop codons, than by those that preserve reading frame. This dependence on reading frame causes these models to systematically miss many poison exons, a clinically relevant category of exons where reading frame is disrupted. This dependence is much weaker in "grey-box" models that build in splicing-specific structure. Our results imply that widely-used black-box models of splicing rely on information that is not accessible to the RNA splicing machinery. Code is available at https://github.com/kavigupta/frame-alignment-checks.

bioinformatics↗

bioRxiv2025

RNA Dynamics Regulate Transcriptional Condensate Vivacity to Drive Gene Coordination

Transcriptional condensates (TCs), enriched with Mediator, orchestrate super-enhancer (SE)-driven gene expression critical for cell identity. However, how their dynamic physical properties shape transcriptional activities remains unclear. Here, we reveal a previously unknown regulatory axis wherein dynamic features of enhancer RNA (eRNA), transcribed but rapidly degraded by the RNA exosome, maintain optimal TC fluidity and stability. Depletion of RNA exosome disrupts TC integrity, altering Mediator and Pol II colocalization, in embryonic stem cells. This TC perturbation attenuates proper transcriptional regulator loading and pause-release regulation, enhances transcriptional noise, and diminishes coordinated eRNA-mRNA expression for SE-associated genes. Multi-omics analyses, live-cell imaging, and computational modeling collectively demonstrate that RNA turnover modulates TC vivacity, facilitating widespread chromatin contacts between SE-containing active A compartments and coordinated transcriptional bursts across SE-associated genes. Our findings establish RNA-dependent condensate dynamics as an essential quality-control mechanism that fine-tunes global transcriptional coordination, maintaining cellular homeostasis and cell identity. HighlightsO_LIRNA exosome is required for the homeostasis of transcriptional condensates (TCs) C_LIO_LIRNA synthesis and decay optimize the fluidity and dynamic properties of TCs C_LIO_LITCs maintain the transcriptional consistency of super-enhancer (SE) genes C_LIO_LITCs support contacts of SE-containing A compartments for coordinated transcription C_LI

molecular biology↗

bioRxiv2024

Mutant p53 Exploits Enhancers to Elevate Immunosuppressive Chemokine Expression and Impair Immune Checkpoint Inhibitors in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective treatments. It is characterized by activating KRAS mutations and p53 alterations. However, how these mutations dysregulate cancer-cell-intrinsic gene programs to influence the immune landscape of the tumor microenvironment (TME) remains poorly understood. Here, we show that p53R172H establishes an immunosuppressive TME, diminishes the efficacy of immune checkpoint inhibitors (ICIs), and enhances tumor growth. Our findings reveal that the upregulation of the immunosuppressive chemokine Cxcl1 mediates these pro-tumorigenic functions of p53R172H. Mechanistically, we show that p53R172H associates with the distal enhancers of the Cxcl1 gene, increasing enhancer activity and Cxcl1 expression. p53R172H occupies these enhancers in an NF-{kappa}B-pathway-dependent manner, suggesting NF-{kappa}Bs role in recruiting p53R172H to the Cxcl1 enhancers. Our work uncovers how a common mutation in a tumor-suppressor transcription factor appropriates enhancers, stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/609802v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b2354dorg.highwire.dtl.DTLVardef@1c96300org.highwire.dtl.DTLVardef@829f26org.highwire.dtl.DTLVardef@19e7d2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Engineered prime editors with minimal genomic errors

Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3 ends. These edited 3 new strands must displace competing 5 strands to install edits, yet a bias toward retaining the competing 5 strands hinders efficiency and can cause indel errors. Using rational design of the constituent Cas9-nickase to reposition prime editor nicks, we discovered that competing 5 strands are destabilized to favor the edited 3 new strands. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next- generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 465:1. One Sentence SummaryPrime editors designed with repositioned DNA breaks nearly eliminate undesired genome editing errors

bioengineering↗

bioRxiv2023

Single-cell nascent RNA sequencing using click-chemistry unveils coordinated transcription

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1-5. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations6-9. However, fundamental questions in the temporal regulation of transcription and enhancer-gene synchrony remain unanswered primarily due to the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq - a novel single-cell nascent RNA sequencing assay using click-chemistry - and unveil the coordinated transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription, and estimates burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells. It reveals the co-transcription of functionally related genes and leverages the replication-dependent non-polyadenylated histone genes transcription to elucidate cell-cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq identifies networks of enhancers and genes and indicates that the bursting of transcription at super-enhancers precedes the burst from associated genes. By imparting insights into the dynamic nature of transcription and the origin and propagation of transcription signals, scGRO-seq demonstrates its unique ability to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.

genomics↗

bioRxiv2023

Wild edible yams from Madagascar: New insights into nutritional composition support their use for food security and conservation

Yams (Dioscorea species) are an important food resource in Madagascar, where both cultivated winged yam (D. alata) and wild edible yams are consumed. However, there is limited knowledge on the nutrient composition of wild edible yams in Madagascar, and on how they compare with the cultivated winged yam. Therefore, in this study, nine wild edible yam species, one with two subspecies from Madagascar (D. bako, D. buckleyana, D. irodensis, D. maciba, D. orangeana, D. pteropoda, D. sambiranensis subsp. bardotiae and subsp. sambiranensis, D. seriflora and Dioscorea species Ovy valiha), were analysed for their nutrient composition, compared with cultivated D. alata. They include 6/6 of the most favoured wild edible yam species in Madagascar. We present new nutrient composition data (protein, carbohydrate/starch, energy, lipid, {beta}-carotene, minerals) for these nine wild edible yam species and show that they contain comparable levels of lipids and starch to D. alata, but none are better sources of protein than D. alata. We show that D. irodensis contains a significantly higher {beta}-carotene content when compared to all other edible yams analysed, and that D. buckleyana, D. irodensis and D. sambiranensis subsp. bardotiae have a higher calcium content than cultivated D. alata, while all nine wild edible yam species analysed contain a higher iron content, compared to cultivated D. alata. The nutrient composition data presented could provide new incentives to conserve wild edible yams and inform on strategies to select Disocorea species for sustainable cultivation and use, providing opportunities to enhance future food security in Madagascar.

plant biology↗

bioRxiv2022

Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and non-dividing cells. These findings establish a new paradigm of targeted nucleases custom-designed for specific mutational applications. TeaserCRISPR-Cas9 can be designed to make otherwise infrequent precise editing pathways dominant in dividing and non-dividing cells

bioengineering↗

bioRxiv2022

A two-gene strategy increases the iron and zinc concentration of wheat flour and improves mineral bioaccesibility for human nutrition.

Dietary deficiencies of iron and zinc cause human malnutrition globally, which can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success so far due to relatively low genetic variation. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat vacuolar iron transporter gene TaVIT2-D with constitutive expression of the rice nicotianamine synthase gene OsNAS2 has the potential to dramatically improve mineral micronutrient intake from wheat products. In two distinct bread wheat cultivars, we show that the VIT-NAS construct led to a two-fold increase in zinc to [~]50 {micro}g g-1 in wholemeal flour and a two-fold increase in both zinc and iron in hand-milled white flour. In highly pure, roller-milled white flour, the concentration of iron was enhanced three-fold to [~]25 {micro}g g-1. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines was associated with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. We conclude that the effects of each gene cassette are additive in altering the total concentration and distribution of iron and zinc in wheat grains. This demonstrates the potential of a transgenic approach to enhance the nutritional quality of wheat well beyond what is possible by breeding approaches in order to alleviate dietary mineral deficiencies.

plant biology↗

bioRxiv2019

CDK13 Mutations Drive Melanoma via Accumulation of Prematurely Terminated Transcripts

Transcriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

biophysics↗

bioRxiv2019

Imprinted maternally-expressed microRNAs antagonize paternally-driven gene programs in neurons

Imprinted genes with parental-biased expression are hypothesized to result from an evolutionary conflict between the parental genomes over procurement of maternal resources. Accordingly, imprinted genes are enriched in pathways regulating nutrient acquisition, energy homeostasis, and growth. Here, we functionally characterize a large cluster of maternally-expressed microRNAs (miRNAs) to explore why they evolved imprinted expression in neurons. Using an induced neuron (iN) culture system, we show maternally-expressed miRNAs from the miR-379/410 cluster repress paternally-expressed genes, including known regulators of energy homeostasis Plagl1 and Peg3. Additional non-imprinted metabolic regulators are also co-targeted by miR-379/410. Maternal deletion of this imprinted miRNA cluster results in de-repression of its targets and up-regulation of a broader gene program regulating feeding behavior and synaptic transmission. These data suggest non-coding RNAs actively engage in parental genomic conflict, whereby maternally-expressed miRNAs antagonize paternally-driven gene programs in neurons.

molecular biology↗

bioRxiv2019

Networks of enhancers and microRNAs drive variation in cell states

Cell-to-cell variation in gene expression is a common feature of developmental processes. Yet, it remains unclear whether molecular mediators can generate variation and how this process is coordinated across loci to allow the emergence of new cell states. Using embryonic stem cells (ESCs) as a model of development, we found interconverting cell states that resemble developmental expression programs and vary in activity at specific enhancers, such as those regulating pluripotency genes Nanog and Sox2 but not Pou5f1 (Oct4). Variable enhancers drive expression of variable genes, including those encoding microRNAs (miRNAs). Notably, variable miRNAs increase cell-to-cell variation by acting on neighborhoods of pluripotency genes. The encoded, variable pluripotency factors bind variable enhancers, forming a feedback loop that amplifies variation and allows the emergence of new cell states. These findings suggest gene regulatory networks composed of enhancers, protein-coding genes, and miRNAs harness inherent variation into developmental outcomes.

cell biology↗

bioRxiv2018

Enhancer features that drive formation of transcriptional condensates

Enhancers, DNA elements that regulate gene expression, contain transcription factor (TF) binding sites. TFs bind short sequence motifs that are present throughout the genome at much higher frequency than active enhancers, and so the features that define active enhancers are not well understood. We show that DNA elements with TF binding site valency, density, and binding affinity above sharply defined thresholds can recruit TFs and coactivators in condensates by the cooperative process of phase separation. We demonstrate that weak cooperative interactions between IDRs of TFs and coactivators in combination with specific TF-DNA interactions are required for forming such transcriptional condensates. IDR-IDR interactions are relatively non-specific with the same molecular interactions shared by many TFs and coactivators, and phase separation is a universal cooperative mechanism. Therefore, whether a genomic locus is an enhancer that can assemble a transcriptional condensate is determined predominantly by its cognate TFs binding site valency and density.

molecular biology↗