Biology subjects

Kouzarides, T.

Publications and source records attributed to Kouzarides, T..

10 recordsLinked to original sources

bioRxiv2025

Compensatory tRNA Modification by DUS3L Confers Resistance to METTL1 Loss in Oesophageal Cancer

tRNA N-methylguanosine (mG46), installed by the METTL1/WDR4 methyltransferase, stabilizes the tRNA pool supporting high proliferative rates. Accordingly, many cancer cell lines are vulnerable to METTL1 loss, underscoring its potential as a therapeutic target. We identify a subset of cell lines insensitive to METTL1 depletion and use oesophageal lines as a model to explore the mechanism. The METTL1-resistant adenocarcinoma line OE33 preserves tRNA balance upon mG46 deficiency by elevating dihydrouridylation at position 47 (D47). Depletion of DUS-L enzymes that catalyse D16/D17/D20/D47 on tRNAs sensitises OE33 to METTL1 loss. Concomitant loss of dihydrouridylation and mG46 destabilizes the canonical L-shaped tRNA structure, disrupts coaxial helix stacking and anticodon-loop presentation, and reduces cellular fitness. Notably, METTL1-sensitive cell lines lack this compensatory pathway due to the deleterious effects of increasing their intrinsically low DUS3L levels. These findings reveal a DUS-L-mediated buffering mechanism that supports tRNA homeostasis in METTL1-resistant cancers and highlight DUS3L abundance as a potential biomarker for predicting METTL1 sensitivity. Moreover, they suggest that combined targeting of DUS-L and METTL1 may provide a therapeutic strategy for overcoming resistance in cancers, exemplified in cell lines such as OE33. HighlightsO_LIThe oesophagus cancer line OE33 shows no proliferative defect upon METTL1/WDR4 loss, despite complete lack of m7G46 on its tRNA. C_LIO_LIOE33 coordinates codon usage and abundance of cognate tRNAs in the absence of METTL1/WDR4; the METTL1/WDR4 sensitive line, OE21, fails to do so. C_LIO_LIDepleting dihydrouridine synthases (DUS-L) renders OE33 sensitive to METTL1/WDR4 loss. C_LIO_LIOE33 responds to lack of METTL1/WDR4 activity by upregulating DUS3L and its activity (D47) on tRNA; the sensitive line OE21 fails to do so. C_LIO_LIMETTL1-sensitive lines have intrinsically low DUS3L and cannot sustain higher levels. C_LIO_LIDUS3L reveals as a potential biomarker for METTL1 sensitivity. C_LI

biochemistry↗

bioRxiv2025

METTL9 sustains vertebrate neural development primarily via non-catalytic functions

METTL9 is an enzyme catalysing N1-methylation of histidine residues (1MH) within eukaryotic proteins. Given its high expression in vertebrate nervous system and its potential association with neurodevelopmental delay, we dissected Mettl9 role during neural development. We generated three distinct mouse embryonic stem cell lines: a complete Mettl9 knock-out (KO), an inducible METTL9 Degron and a line endogenously expressing a catalytically inactive protein, and assessed their ability to undergo neural differentiation. In parallel, we down-regulated mettl9 in Xenopus laevis embryos and characterised their neural development. Our multi-omics data indicate that METTL9 exerts a conserved role in sustaining vertebrate neurogenesis. This is largely independent of its catalytic activity and occurs through modulation of the secretory pathway. METTL9 interacts with key regulators of cellular transport, endocytosis and Golgi integrity; moreover, in Mettl9KO cells Golgi becomes fragmented. Overall, we discovered the first developmental function of Mettl9 and linked it to a 1MH-independent pathway, namely, the maintenance of the secretory system, which is essential throughout neural development.

developmental biology↗

bioRxiv2024

PhOxi-seq detects enzyme-dependent m2G in multiple RNA types

In recent years, RNA-modifying enzymes have gained significant attention due to their impact on critical RNA-based processes, and consequently human pathology. However, identifying sites of modifications throughout the transcriptome remains challenging largely due to the lack of accurate and sensitive detection technologies. Recently, we described PhOxi-seq as a method capable of confirming known sites of m2G within abundant classes of RNA, namely purified rRNA and purified tRNA. Here, we further explore the selectivity of PhOxi-seq and describe an optimised PhOxi-seq workflow, coupled to a novel bioinformatic pipeline, that is capable of detecting enzyme-dependent m2G sites throughout the transcriptome, including low abundant mRNAs. In this way, we generated the first database of high confidence sites of THUMPD3-dependent m2G in multiple RNA classes within a human cancer cell line and further identify non-THUMPD3 controlled sites throughout the transcriptome.

biochemistry↗

bioRxiv2023

Pseudouridine guides germline small RNA transport and epigenetic inheritance

Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes1. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation2,3. In C. elegans, these inherited small RNAs have poly (UG) tails4, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine ({Psi}) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect {Psi} in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with {Psi} and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, {Psi} has a conserved role in marking inherited small RNAs in the germline. One-Sentence SummaryPseudouridine marks germline small RNAs in plants and mammals, impacting epigenetic inheritance via nuclear transport.

genetics↗

bioRxiv2021

Predicting genes associated with RNA methylation pathways using machine learning

RNA methylation plays an important role in functional regulation of RNAs, and has thus attracted an increasing interest in biology and drug discovery. Here, we collected and collated transcriptomic, proteomic, structural and physical interaction data from the Harmonizome database, and applied supervised machine learning to predict novel genes associated with RNA methylation pathways in human. We selected five types of classifiers, which we trained and evaluated using cross-validation on multiple training sets. The best models reached 88% accuracy based on cross-validation, and an average 91% accuracy on the test set. Using protein-protein interaction data, we propose six molecular sub-networks linking model predictions to previously known RNA methylation genes, with roles in mRNA methylation, tRNA processing, rRNA processing, but also protein and chromatin modifications. Our study exemplifies how access to large omics datasets joined by machine learning methods can be used to predict gene function.

bioinformatics↗

bioRxiv2020

Identification of SARS-CoV-2 induced pathways reveal drug repurposing strategies

The global outbreak of SARS-CoV-2 necessitates the rapid development of new therapies against COVID-19 infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein (SIP) network, based on disease signatures defined by COVID-19 multi-omic datasets(Bojkova et al., 2020; Gordon et al., 2020), and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials(Clinicaltrials.gov, 2020) testifying to the validity of the approach. Using artificial neural network analysis we classified these 200 drugs into 9 distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (130) and immune response (70). A subset of drugs implicated in viral replication were tested in cellular assays and two (proguanil and sulfasalazine) were shown to inhibit replication. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.

pharmacology and toxicology↗

bioRxiv2019

RNA modifications detection by comparative Nanopore direct RNA sequencing

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. To date, over 150 naturally occurring PTMs have been identified, however the overwhelming majority of their functions remain elusive. In recent years, a small number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing (DRS) technology has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework to evaluate the presence of modifications in DRS data. To do so, we compare an RNA sample of interest against a non-modified control sample. Our strategy does not require a training set and allows the use of replicates to model biological variability. Here, we demonstrate the ability of Nanocompore to detect RNA modifications at single-molecule resolution in human polyA+ RNAs, as well as in targeted non-coding RNAs. Our results correlate well with orthogonal methods, confirm previous observations on the distribution of N6-methyladenosine sites and provide novel insights into the distribution of RNA modifications in the coding and non-coding transcriptomes. The latest version of Nanocompore can be obtained at https://github.com/tleonardi/nanocompore.

genomics↗

bioRxiv2019

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role of Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interact with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but might rather regulate other processes in the nucleoplasm. Summary blurbSox2 interacts with RNA-binding proteins and diverse RNAs

molecular biology↗

bioRxiv2018

A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry

The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Next-generation sequencing approaches are generally unable to capture modifications, although workarounds for some epigenetic marks exist. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. In particular, existing software solutions lack the raw performance and statistical grounding to efficiently handle the large variety of modifications present on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in three original datasets of varying complexity. In a human tRNA sample, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.

bioinformatics↗

bioRxiv2017

A lncRNA/Lin28/Let7 Axis Coupled To DNA Methylation Fine Tunes The Dynamics Of A Cell State Transition

Execution of pluripotency requires progression from the naive status represented by mouse embryonic stem cells (ESCs) to a condition poised for lineage specification. This process is controlled at transcriptional, post-transcriptional and epigenetic levels and non-coding RNAs are contributors to this regulation complexity. Here we identify a molecular cascade initiated by a long non-coding RNA (lncRNA), Ephemeron (Epn), that modulates the dynamics of exit from naive pluripotency. Epn deletion delays the extinction of ESC identity, an effect mediated by perduring expression of the pivotal transcription factor Nanog. In the absence of Epn, Lin28a expression is reduced, resulting in an elevated level of Mirlet7g that suppresses de novo methyltransferases Dnmt3a/b. Dnmt3a/b deletion also retards exit from the ESC state, and is associated with delayed promoter methylation and slower down-regulation of Nanog. Altogether, our findings reveal a lncRNA/miRNA/DNA methylation axis that facilitates a timely stem cell state transition.

cell biology↗