Biology subjects

Kus, K.

Publications and source records attributed to Kus, K..

15 recordsLinked to original sources

bioRxiv2026

Adulthood depletion of Integrator extends lifespan and healthspan via defective pre-mRNA processing

Identifying strategies to mitigate age-related physiological decline remains a central challenge. During ageing, the transcriptome undergoes extensive remodelling, but how this affects organismal health and lifespan is not well understood. The Integrator complex plays a central role in RNA polymerase II transcription and RNA 3' end processing. Surprisingly, we find that depletion of most Integrator subunits specifically in adults extends lifespan and healthspan in the nematode C. elegans. We show that loss of the catalytic subunit INTS-11 disrupts 3' end formation of small nuclear and spliced leader RNAs, impairing trans-splicing and promoting outron retention in a subset of transcripts enriched for spliceosomal and nucleocytoplasmic transport genes. These RNA-processing defects lead to altered levels of endogenous siRNAs, which are required for the longevity and healthspan benefits of INTS-11 depletion. In parallel, outron retention disrupts nuclear-encoded mitochondrial gene expression and protein production, inducing mitochondrial dysfunction and promoting lifespan extension. We also demonstrate that loss of INTS-11 perturbs transcription elongation at genes where Integrator is present at promoters, and that upregulation of enhancer elements within intragenic regions can affect the expression and isoform usage of nearby genes. Together, our findings identify Integrator as a key upstream regulator of non-coding RNA transcription, which in turn impacts protein-coding gene expression and mitochondrial function to shape the ageing process.

cell biology↗

bioRxiv2026

Mature tumoroids recapitulate clinically relevant drug response through extended 3D culture in PDAC

BackgroundDrug responses in pancreatic ductal adenocarcinoma (PDAC) vary sharply across in vitro culture formats, but most 2D-3D comparisons conflate microenvironmental cues with time-dependent cellular adaptation. As a result, conventional assays frequently overestimate drug efficacy and poorly reflect clinical pharmacology. Main findingsWe profiled MiaPaCa-2, PANC-1, and CFPAC-1 grown in an extracellular-matrix (ECM) hydrogel for 1-12 days, defining extended 3D cultures ([≥]10 days) as mature tumoroids, and quantified 72 h drug responses to a multi-class oncology panel using growth-rate (GR) metrics to normalize for proliferation across formats and durations. Prolonged 3D pre-culture induced broad tolerance, with typical 10-100x reductions in sensitivity to standards of care (5-fluorouracil, SN38, oxaliplatin, gemcitabine, paclitaxel), following a reproducible susceptibility hierarchy (MiaPaCa-2 > PANC-1 > CFPAC-1) after GR correction. In mature tumoroids, GR values closely approximated clinically observed plasma exposures (e.g., within <4x for 5-FU and <0.5x for gemcitabine), whereas 2D and short-term organoid assays markedly underestimated resistance, often by >100x, thereby overstating drug activity. Notably, CFPAC-1 exhibited increased sensitivity to SN38 and trametinib under mature-organoid conditions, demonstrating that microenvironmental conditioning can invert responses for selected mechanisms. Transcriptomic profiling revealed coordinated up-regulation of multiple ABC transporters with extended 3D residence, tracking resistance phenotypes across lines and implicating transporter-linked tolerance programs. SignificanceTogether, these data identify time-in-3D and the emergence of mature tumoroids as dominant, previously under-controlled determinants of PDAC pharmacology that both induce tolerance and unmask context-dependent vulnerabilities. We propose incorporating both short-term and mature-tumoroid screening arms into preclinical workflows, reporting pre-culture duration alongside GR-normalized effect sizes, and leveraging transporter-informed biomarkers to guide regimen prioritization and sequencing. This framework enhances physiological relevance, reproducibility, and translational fidelity in PDAC drug discovery.

cancer biology↗

bioRxiv2025

The PP1 phosphatase complex coordinates pre-mRNA 3end processing and termination of RNA polymerase II transcription

Phosphorylation plays a central role in coordinating transcription with pre-mRNA maturation, yet the mechanistic understanding of how transcription is regulated by phosphorylation remains limited. The PP1 phosphatase removes CDK9-dependent phosphorylation of Pol II and Spt5, triggering transcription termination. Here, we demonstrate that PNUTS enhances PP1 activity, whereas WDR82 mediates an interaction between PP1 and the pre-mRNA 3' end processing machinery subunit Symplekin. We present the first structure of a PNUTS-WDR82-Symplekin-Ssu72 complex, revealing that PNUTS and Symplekin bind WDR82 through distinct interfaces using conserved short linear motifs. Mutations that inactivate PP1s catalytic activity or disrupt its interaction with either PNUTS or the 3' end processing machinery led to impaired transcription elongation, inefficient cleavage of nascent transcripts, altered poly(A) site selection, and widespread transcription termination defects. Our findings demonstrate that the PP1 complex is essential for coupling transcription to pre-mRNA 3' end maturation, ensuring production of functional mRNA.

molecular biology↗

bioRxiv2024

Conserved protein Seb1 that interacts with RNA polymerase II and RNA is a bona fide transcription elongation factor

Maturation of protein-coding precursor messenger RNA (pre-mRNA) is closely linked to RNA polymerase II (Pol II) transcription. However, the mechanistic understanding of how RNA processing is coordinated with transcription is incomplete. Conserved proteins interacting with the C-terminal domain of the largest catalytic subunits of Pol II and nascent RNA (CID-RRM factors) were demonstrated to play a role in mRNA 3-end processing and termination of Pol II transcription. Here, we employ a fully reconstituted system to demonstrate that fission yeast CID-RRM factor Seb1 acts as a bona fide elongation factor in vitro. Our analyses show that Seb1 exhibits context-dependent regulation of Pol II pausing, capable of either promoting or inhibiting pause site entry. We propose that CID-RRM factors coordinate Pol II transcription and RNA 3-end processing by modulating the rate of Pol II transcription.

molecular biology↗

bioRxiv2024

Development of antisense tools to study Bodo saltans and its intracellular symbiont

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here we investigated the potential for using antisense inhibition to analyse host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the B. saltans and its symbiont, as the holobiont feeds on the antisense molecules - and increases in numbers - upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols which can be used to study the biology of this microeukaryote and its microbial associates.

molecular biology↗

bioRxiv2024

Mitochondria-targeted hydrogen sulfide donor reduces atherogenesis by reprogramming macrophages and increasing UCP1 expression in vascular smooth muscle cells

AimsAtherosclerosis is a leading cause of morbidity and mortality in the Western countries. A growing body of evidence points to the role of mitochondrial dysfunction in the pathogenesis of atherosclerosis. Recently, it has been shown that mitochondrial hydrogen sulfide (H2S) can complement the bioenergetic role of Krebs cycle leading to improved mitochondrial function. However, controlled, direct delivery of H2S to mitochondria was not investigated as a therapeutic strategy in atherosclerosis. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with mitochondrial H2S donor AP39 on the development of atherosclerotic lesions in apolipoprotein E knockout (apoE-/-) mice. ResultsOur results indicated that AP39 reduced atherosclerosis in apoE-/- mice and stabilized atherosclerotic lesions through decreased total macrophage content and increased collagen depositions. Moreover, AP39 reprogrammed macrophages from proinflammatory M1 to anti-inflammatory M2 in atherosclerotic lesions. It also upregulated pathways related to mitochondrial function, such as cellular respiration, fatty acid {beta}-oxidation and thermogenesis while downregulated pathways associated with immune system, platelet aggregation and complement and coagulation cascades in the aorta. Furthermore, treatment with AP39 increased the expression of mitochondrial brown fat uncoupling protein 1 (UCP1) in vascular smooth muscle cells (VSMCs) in atherosclerotic lesions and upregulated mRNA expression of other thermogenesis-related genes in the aorta but not perivascular adipose tissue (PVAT) of apoE-/- mice. Finally, AP39 treatment decreased markers of activated endothelium and increased endothelial nitric oxide synthase (eNOS) expression and activation. ConclusionsTaken together, mitochondrial H2S donor AP39 could provide potentially a novel therapeutic approach to the treatment/prevention of atherosclerosis.

pharmacology and toxicology↗

bioRxiv2024

Mitochondria-targeted hydrogen sulfide donor reduces fatty liver and obesity in mice fed a high fat diet by inhibiting de novo lipogenesis and inflammation via mTOR/SREBP-1 and NF-κB signaling pathways

BackgroundMetabolic diseases that include obesity and metabolic-associated fatty liver disease (MAFLD) are a rapidly growing worldwide public health problem with unmet clinical need. The pathogenesis of MAFLD is very complex including abnormally increased lipogenesis, chronic inflammation, mitochondrial dysfunction, and oxidative stress. A growing body of evidence suggests that hydrogen sulfide (H2S) is an important player in the liver, impacting lipid metabolism and mitochondrial function. However, direct delivery of H2S to mitochondria has not been investigated as a therapeutic strategy in obesity-related metabolic disorders. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with a mitochondria sulfide delivery molecule (AP39) on the development of fatty liver and obesity in a high fat diet (HFD) fed mice. ResultsOur results demonstrated that AP39 reduced fatty liver in HFD-fed mice, which was corresponded with decreased triglyceride content in the liver and plasma as well as increased GSH/GSSG ratio in the plasma. Furthermore, treatment with AP39 downregulated pathways related to biosynthesis of unsaturated fatty acids, lipoprotein assembly and PPAR signaling in the liver of HFD-fed mice. It also led to a decrease in de novo lipogenesis in the liver by downregulating mTOR/SREBP-1/SCD1 signaling pathway. Moreover, AP39 administration alleviated obesity in HFD-fed mice, which was reflected by reduced weight of mice and adipose tissue, decreased leptin levels in the plasma and upregulated expression of ATGL, a lipolysis enzyme in epididymal white adipose tissue (eWAT). Finally, AP39 reduced inflammation in the liver and eWAT measured as the expression of pro inflammatory markers (Il1b, Il6, Tnf, Mcp1), which was due to the downregulation of mTOR/NF-{kappa}B signaling pathway. ConclusionsTaken together, mitochondria-targeted sulfide delivery molecules could potentially provide a novel therapeutic approach to the treatment/prevention of obesity-related metabolic disorders.

molecular biology↗

bioRxiv2024

Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro. Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.

cancer biology↗

bioRxiv2024

3D culture of pancreatic cancer cells in vitro recapitulates an aberrant mitochondrial oxidative phosphorylation genotype observed in vivo

The treatment of many aggressive cancers remains a significant unmet medical need. An aberrant dependence of tumors on mitochondrial oxidative phosphorylation pathways has been well characterized in mediating the progression of pancreatic cancers, as well as some other malignancies. However, the discovery and development of new therapeutic strategies targeting or manipulating this pathway in pancreatic tumors has been relatively slow when compared with other cancers, limiting the current options for treating patients. One key technical challenge in discovering new therapies has been the limited ability of cancer cell lines when grown in 2D conditions to recapitulate the mitochondrial oxidative phosphorylation pathways in vitro that have been observed in vivo. In part, this is because 2D cultures fail to mimic the extracellular 3D tumoral environment. More generally, the translation of data based on 2D pancreatic cancer cell culture systems have also been constraining options for discovering new therapies. Conversely, 3D cultures can provide an improved technology platform in which pathologically relevant pathways in tumors can be recapitulated and analyzed in vitro. In this study, we have demonstrated in 3D cultures the reconstruction of the mitochondrial oxidative phosphorylation genotype in vitro, which more closely resembles that observed in vivo. Our study highlights the value of using transcriptomic readouts as a method to demonstrate the relevance and utility of 3D preclinical models in vitro, when grown in a more physiological extracellular environment. This key technological advance can now better enable the discovery and subsequent development of new therapeutic strategies targeting disease-relevant pathways found in pancreatic and other tumors.

cancer biology↗

bioRxiv2023

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts.

The timely termination of RNA polymerase II (Pol II) transcription is critical for polymerase recycling and preventing interference with the expression of the neighbouring genes. Termination of Pol II transcription involves exoribonucleolytic decay of the nascent RNA by 5-3 exonuclease Xrn2. Xrn2 attacks the 5-PO4-end and executes degradation of the nascent RNA generated by the endonucleolytic cleavage at the poly(A) site which eventually leads to Pol II release from DNA. However, the molecular details of when and how during this process Xrn2 interacts with the Pol II elongation complex to mediate its dissociation from DNA is not understood. Here, we demonstrate that Xrn2 interacts with Pol II and Spt5, a conserved transcription factor that controls Pol II processivity and pausing. Importantly, Xrn2 activity is stimulated by Spt5 in vitro and Spt5-depleted cells show defective transcription termination. Our results support a model where Xrn2 first forms a stable complex with the elongating Pol II to acquire its full activity in degrading nascent RNA. Spt5 also promotes premature termination attenuating the expression of non-coding transcripts. By contrast, Spt5 depletion leads to Pol II retention at promoters of protein-coding genes. Pol IIs that transcribe into a gene body in the absence of Spt5 exhibit severely reduced elongation rates and defective pre-mRNA processing. We propose that Spt5 plays a major role in the production of functional mRNA by directly stimulating the activity of the RNA enzymes and preventing entry of Pol II complexes not configured to support transcription and pre-mRNA processing into elongation.

molecular biology↗

bioRxiv2022

Thermodynamic analysis of Zα domain-nucleic acid interactions

DNA/RNA molecules adopting the left-handed conformation (Z-form) have been attributed with immunogenic properties. However, their biological role and importance has been a topic of debate for many years. The discovery of Z-DNA/RNA binding domains (Z domains) in varied proteins that are involved in the innate immune response, such as the interferon inducible form of the RNA editing enzyme ADAR1 (p150), Z-DNA binding protein 1 (ZBP1), the fish kinase PKZ and the poxvirus inhibitor of interferon response E3L, indicates important roles of Z-DNA/RNA in immunity and self/non-self-discrimination. Such Z domain-containing proteins recognise left-handed Z-DNA/RNA in a conformation-specific manner. Recent studies have implicated these domains in virus recognition. Given these important emerging roles for the Z domains, it is pivotal to understand the mechanism of recognition of the Z-DNA/Z-RNA by these domains. To this end, we assessed the binding thermodynamics of Z domain from ORF112 and ADAR1 on T(CG)3 and T(CG)6 oligonucleotides which have high propensity to adopt the Z-conformation. Our study highlights important differences in the mode of binding by the two Z domains originating from different proteins. Site-directed mutagenesis was employed together with isothermal titration calorimetry to tease apart finer details of the binding thermodynamics. Our work advances the understanding on binding thermodynamics of Z domains to their cognate nucleic acid substrates and contributes to the efforts to gain a complete appreciation of this process.

biochemistry↗

bioRxiv2021

Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind nucleic acids

Z domains are a subfamily of winged Helix-Turn-Helix (wHTH) domains found exclusively in proteins involved in the nucleic acids sensory pathway of vertebrate innate immune system and host evasion by viral pathogens. Interestingly, they are the only known protein domains that recognise the left-handed helical conformation of both dsDNA and dsRNA, known as Z-DNA and Z-RNA. Previously, it has been demonstrated that ADAR1 and ZBP1, two proteins possessing the Z domains, localize to cytosolic stress granules. It was further speculated that such localization is principally mediated by Z domains. To characterize and better understand such distinct and specific localization, we characterised the in vivo interactions and localization pattern for the amino terminal region of human DAI harbouring two Z domains (Z{beta}DAI). Using immunoprecipitation and mass spectrometry, we identified several interacting partners that were components of the complex formed by Z domains and RNAs. Differential interacting partners to wild-type Z, relative to mutant proteins, demonstrated that most of the physiologically relevant interactions are mediated by the nucleic acid binding ability of the Z{beta}. Further, we also show enrichment of selected complex components in cytoplasmic stress granules under conditions of stress. This ability is mostly lost in the mutants of Z{beta}DAI (Z{beta}DAI 4xmut) that lack nucleic-acid binding ability. Thus, we posit that the mechanism for the translocation of Z domain-containing proteins to stress granules is mainly mediated by the nucleic acid binding ability of their Z domains. Finally, we demonstrate that FUS and PSF/p54nrb, two RNA binding proteins with established roles in stress granules, interact with Z, which provides strong evidence for a role of these proteins in the innate immune system.

biochemistry↗

bioRxiv2020

Comparative poly(A)+ RNA interactome capture of fission yeast RNA exosome mutants reveals insights into RNA biogenesis

The nuclear RNA exosome plays a key role in mediating degradation and processing of multiple cellular RNAs. Recognition of the specific RNA substrates by the exosome is mediated by the RNA-binding co-factors. Transient binding of co-factors either to the exosome or the substrate RNAs as well as their rapid decay make identification of the co-factors challenging. Here, we employ a comparative poly(A)+ RNA interactome capture approach in the fission yeast exosome mutants to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identified multiple proteins whose occupancy on RNA is altered in the exosome mutants including zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of the exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

genomics↗

bioRxiv2019

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA-binding activity and provide key information about the RNA-binding properties of large multi-protein complexes, such as the mRNA 3 end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

molecular biology↗

bioRxiv2017

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

molecular biology↗