Biology subjects

Inga, A.

Publications and source records attributed to Inga, A..

13 recordsLinked to original sources

bioRxiv2026

Allele-resolved monosome and polysome sequencing identifies functional cis-acting variants affecting mRNA translation efficiency

To prioritize germline genetic variants affecting mRNA fate at the post-transcriptional and translational levels, we leveraged sucrose-gradient-based isolation of 80S monosomes and polysomes, followed by mRNA retrieval and paired-end sequencing. Total cytoplasmic RNA was also sequenced for comparison. Experiments were performed in the non-transformed cell line RPE-1, cultured under basal conditions or upon p53 activation by Nutlin. Differential gene expression analysis confirmed a canonical p53 response. Heterozygous SNPs and SNVs were identified from the RNA-seq data, and allelic fractions (AF) were calculated for total, monosomal, and polysomal mRNAs. Variants showing reproducible AF differences across fractions beyond experimental variability were defined as tranSNPs. Among nearly 7000 heterozygous variants analyzable in polysomal or total RNA and over 5000 in monosomal mRNA, 1155 displayed a significant imbalance. Reporter assays performed in both RPE-1 and HCT116 cells validated allelic or haplotype effects for 17 selected variants in UTRs and coding regions, confirming differences in 15 cases, with evidence of cell line-specific responses. Proteomic analysis further supported allelic imbalance for selected missense variants. Overall, tranSNPs were identified in a non-transformed cell line at frequencies comparable to those in cancer cells, thereby extending their implications in human physiology. Further, monosome profiling enabled improved detection sensitivity of tranSNPs without positional bias, suggesting that 80S profiling improves detection of allele-specific translational regulation in RPE-1 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/723424v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f10281org.highwire.dtl.DTLVardef@db91ccorg.highwire.dtl.DTLVardef@140ea91org.highwire.dtl.DTLVardef@e80c99_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2026

Centrosome architecture and m6A-dependent gating of p53 surveillanceafter whole-genome doubling

Whole-genome doubling (WGD) generates a tetraploid state that challenges cellular homeostasis and is strongly selected against in TP53-proficient contexts. Although centrosome amplification serves as a structural correlate of genome doubling, how centrosomal features engage and sustain p53 surveillance remains incompletely understood. Here, we develop a genetically encoded MDM2-based fluorescent reporter to monitor Caspase-2 pathway activation in living cells after WGD. Combining pharmacological profiling and genome-wide CRISPR screening, we uncover a multi-layered regulation of p53 responses. We identify PLK1-dependent centriole maturation and subdistal appendage integrity as architectural determinants of PIDDosome-dependent Caspase-2 activation, independent of distal appendage assembly. We show that upon WGD, Caspase-2-mediated cleavage of MDM2 reshapes the p53-MDM2 regulatory loop, enabling sustained p53 signaling. This sustained output depends on the catalytic activity of METTL3 and associated m6A writer proteins. Together, these findings demonstrate that centrosome architecture and epitranscriptomic regulation cooperate to shape p53 surveillance after genome doubling. HighlightsO_LIWhole-genome doubling creates a surveillance state sensed by centrosome cluster architecture. C_LIO_LICaspase-2 activation converts the p53-MDM2 loop from negative to positive feedback. C_LIO_LIm6A methylation establishes competence of the p53 module for sustained output. C_LIO_LIA genetically encoded biosensor enables functional dissection of this circuit. C_LI

cell biology↗

bioRxiv2025

Polysomal profiling coupled to allele-specific proteomics reveals an EIF4H tranSNP allele possessing higher mRNA translation potential.

To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116-derived cell lines, computing allelic fractions in total and polysome-associated RNA from RNA-seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are nonsynonymous. To validate instances of allele-specific translation, a proof-of-concept proteomics approach was developed to quantify the relative expression of pairs of endogenous proteins resulting from the decoding of alleles containing the nonsynonymous heterozygous variants. In particular, the G>A, R183H missense SNV rs1554710467 in the EIF4H gene was investigated. The alternative peptide containing H183 was significantly more abundant than the corresponding one containing R183, consistent with the overrepresentation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subcellular localization. These findings support the classification of rs1554710467 as a gain-of-function allele. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels, exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression control that could underlie inter-individual differences in disease-relevant phenotypes.

genetics↗

bioRxiv2025

A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

Relatively few studies have examined the link between SNPs and mRNA translation, despite the established importance of translational regulation in shaping cell phenotypes. We developed a pipeline analyzing the allelic imbalance in total and polysome-bound mRNAs from paired RNA-seq data of HCT116 cells and identified 40 candidate tranSNPs, i.e. SNPs associated with allele-specific translation. Among them, the SNP rs1053639 (T/A) on DNA damage-inducible transcript 4 (DDIT4) 3UTR was identified, with the reference T allele showing a higher polysome association. rs1053639 TT clones generated by genome editing exhibited significantly higher DDIT4 protein levels than AA ones. The difference in DDIT4 proteins was even greater when cells were treated with Thapsigargin or Nutlin, two perturbations that induce DDIT4 transcription. The RNA-binding protein RBMX influenced these allele-dependent differences in DDIT4 protein expression, as shown by RNA-EMSA, RIP, and smiFISH assays. RBMX depletion reduced DDIT4 protein in TT clones to the AA levels. Functionally, TT clones more effectively repressed mTORC1 under ER stress, while AA clones outcompeted TT clones in vitro or when injected in zebrafish embryos. RBMX depletion increased the fitness of TT cells in co-culture experiments. The rs1053639 AA genotype, under a recessive model, correlates with poor prognosis in TCGA cancer data. Key points- Translatome analysis in HCT116 cells revealed allele-specific mRNA translation for 40 SNPs - rs1053639 (T/A) in DDIT4 3UTR showed allelic differences in mRNA localization & protein expression - AA cells showed weaker mTOR inhibition & higher proliferation; AA individuals had poorer prognosis

molecular biology↗

bioRxiv2023

The presence of a G4 prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast S. cerevisiae

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast S. cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides for basal reporter activity, the P53 RE enables LUC1 transactivation under the control of the human P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family proteins dependent expression was measured after shifting the yeast cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter can increase its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this improved accessibility, when present near the target binding site of P53 family transcription factors can be exploited in order to regulate the transcriptional activity of P53, P63 and P73 proteins.

cancer biology↗

bioRxiv2022

Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation occurring in the population of south Brazil. In this work, we have combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. Besides the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt bridge interaction that stabilizes the p53 tetramerization domain were studied (R337D, D352R, and the double mutation R337D plus D352R). Simulations revealed a destabilizing effect of pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay performed in yeast by tuning the expression of wild-type and mutant p53 proteins revealed that p53 tetramerization mutations could decrease transactivation potential and alter transactivation specificity, in particular, by better tolerating the negative features in weak DNA binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53 conformational stability and function.

cancer biology↗

bioRxiv2021

Novel allosteric mechanism of dual p53/MDM2 and p53/MDM4 inhibition by a small molecule

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising strategy. However, high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which blocks both p53/MDM2 and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA and protoporphyrin IX (PpIX). Ion-mobility mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions. Contribution to the fieldGiven the immense importance of the p53 tumor suppressor for cancer, efforts have been made to identify p53 activators, which sterically inhibit MDM2. Because high-affinity MDM2 inhibitors are facing problems with considerable side effects, other approaches are needed to reactivate p53 for improved cancer therapy. The allosteric mechanism of action of p53 activator RITA, which we discovered, and its dependence on the oncogenic switch, is an unexpected turn in the p53 story. Our findings provide a basis for the development of p53 activators with a similar mode of functioning, either through the classical drug discovery route or through the drug repurposing approach. Allosteric modulators might have great potential as single agents, or in combination with the standard of care. Further, p53 modulators could serve as invaluable tools to better understand its biology.

biochemistry↗

bioRxiv2021

The pseudouridine synthase dyskerin binds to cytoplasmic H/ACA-box snoRNA retaining transcripts affecting nuclear hormone receptor dependence

Dyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. Since its defective function induces specific alterations in gene expression, we sought to unbiasedly identify mRNAs regulated by dyskerin. We found that dyskerin depletion affects the expression or the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin and can interact with cytoplasmic ribosomes. We then characterized the cytoplasmic dyskerin RNA interactome finding both H/ACA box snoRTs and protein-coding transcripts. Since a fraction of these latter transcripts is involved in the nuclear hormone receptor binding, we tested to see if this specific activity is affected by dyskerin. Results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer. Our work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

molecular biology↗

bioRxiv2020

The RNA helicase DHX30 coordinates cytoplasmic translation and mitochondrial function contributing to cancer cell survival

DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, depletion of both DHX30 isoforms exhibits higher global translation but slower proliferation, and reduced mitochondrial energy metabolism. Isoform-specific silencing established a role for cytoplasmic DHX30 in modulating global translation. The impact on global translation and proliferation were confirmed in U2OS and MCF7 cells, although the effect of DHX30 depletion on mitochondrial gene expression was observed only in MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified a gene signature comprising DHX30 and fourteen mitoribosome transcripts that we candidate as direct targets: this signature shows prognostic value in several TCGA cancer types, with higher expression associated with reduced overall survival. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells. Author summaryTranslation occurs in the cell both through cytoplasmic and mitochondrial ribosomes, respectively translating mRNAs encoded by the nuclear and the mitochondrial genome. Here we found that DHX30, an RNA-binding protein implicated in p53-dependent apoptosis, enhances the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing that of the mitoribosome mRNAs when silenced. This coordination of the cytoplasmic and mitochondrial translation machineries affected both cell proliferation and energy metabolism, suggesting an important role for this mechanism in determining the fitness of cancer cells. Indeed, the analysis of publicly available cancer datasets led us to define a 15-genes signature that is able to affect the prognosis of a subset of cancer types. In this subset, we found that higher expression of the genes composing the signature is associated with a worse prognosis. We thus propose DHX30 as a potential vulnerability in cancer cells, that could be targeted to develop novel therapeutic strategies.

molecular biology↗

bioRxiv2019

p53-induced apoptosis is specified by a translation program regulated by PCBP2 and DHX30.

Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits of Nutlin. Here, we report a new translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to be associated with the enhanced translation of mRNAs carrying multiple copies of a newly identified 3UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identified PCBP2 and DHX30 as CGPD-motif interactors. We found that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Thus, upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs is increased, and the response to Nutlin shifts towards apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells, that undergo Nutlin-induced apoptosis, leads to decreased translation of CGPD-motif mRNAs. Overall, this work establishes the role of PCBP2-DHX30 in controlling the translation of CGPD-motif mRNAs and thus provide a new mechanism to modulate the induction of p53-dependent apoptosis.

molecular biology↗

bioRxiv2019

SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy

The contribution of ribosome heterogeneity and ribosome-associated factors to the molecular control of proteomes in health and disease remains enigmatic. We demonstrate that Survival Motor Neuron (SMN) protein, loss of which causes the neuromuscular disease spinal muscular atrophy (SMA), binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are positioned within the first five codons of a set of mRNAs which are enriched in IRES-like sequences in the 5UTR and rare codons at the beginning of their coding sequence. Loss of SMN at early-stages of SMA induces translational defects in vivo, characterized by ribosome depletion in rare codons at the third and fifth position of the coding sequence. These positional defects cause ribosome depletion from mRNAs bound by SMN-primed ribosomes and translational impairment of proteins involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs which encode proteins relevant to SMA pathogenesis.

neuroscience↗

bioRxiv2018

Novel allosteric mechanism of p53 activation by small molecules for targeted anticancer therapy

Given the immense significance of p53 restoration for anti-cancer therapy, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in p53 regulation, whose targeting by RITA, PpIX and licofelone block the binding of p53 inhibitors, MDM2 and MDMX. Deletion and mutation analysis followed by molecular modeling, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators. Our study provides the basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacological potential.

cancer biology↗

bioRxiv2018

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, compensates for p53 loss and induces apoptosis in TP53-deficient cancer cells.

BackgroundThe p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated {Delta}N isoform that inhibits TAp73 and p53 and thus, acts as an oncogene.\n\nResultsBy employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid (ALA) applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch.\n\nConclusionOur findings may in future contribute to the successful repurposing of PpIX into clinical practice.

cancer biology↗