Biology subjects

Britto-Borges, T.

Publications and source records attributed to Britto-Borges, T..

10 recordsLinked to original sources

bioRxiv2025

Genomic G quadruplexes regulate mRNA splicing

Genomic G quadruplexes (G4) are non-canonical DNA structures that regulate gene expression primarily through transcriptional control. Here, we uncover that DNA G quadruplexes are critical determinants of pre-mRNA splicing. G4s on the DNA template strand serve as recruiting elements for the RNA-binding proteins SRSF9 and WBP11 in order to facilitate productive splicing of adjacent pre-mRNAs. This process is controlled by protein arginine methyltransferase 5 (PRMT5) that releases SRSF9 and WBP11 from G4s through arginine methylation. Acyl-CoA dehydrogenase very long-chain specific (ACADVL) is a gene highly regulated by this mechanism since targeting G4 stability prevents mis-splicing and restores ACADVL protein levels. In the heart, deletion of Prmt5 recapitulates defective splicing and results in progressive cardiac failure along with loss of ACADVL in mice. Importantly, we find that Acadvl regulation plays a critical role for cardiomyopathy as restoring Acadvl expression in Prmt5 knockout mice prevents cardiac dysfunction. This study establishes an unanticipated fundamental principle by which genomic G quadruplexes act as splicing enhancers of associated pre-mRNAs, and reveals an essential role in cardiac homeostasis.

cell biology↗

bioRxiv2025

CAMK2D causes heart failure in RBM20 cardiomyopathy

Although heart disease can arise from different etiologies, current treatment is not tailored to the different underlying causes but is rather a one-size-fits-all approach. Importantly, not all patients benefit from this treatment regimen, which means the number needed to treat is very high. Moreover, this makes clinical trials large and costly, limiting clinical translation. Thus, there is a high medical need to develop a first etiology-specific therapy. Mutations in RBM20, a splicing factor that targets multiple pivotal cardiac genes including TTN and CAMK2D, cause a clinically aggressive form of dilated cardiomyopathy (DCM) with a high risk of malignant ventricular arrhythmias. Here, we hypothesized that CAMK2D is the heart disease-causing target of RBM20. We crossed Camk2d- to Rbm20-deficient mice and found that double knockout (DKO) mice were protected from heart failure and sudden cardiac death. Phosphorylation of multiple CAMK2D targets was increased in Rbm20-deficient mouse hearts, which was reverted in DKO hearts, confirming that CAMK2D is not only misspliced but also overactivated. AAV9-mediated re-expression of single CAMK2D splice variants in DKO mice reintroduced cardiac dysfunction irrespective of the splice variant, unmasking that overactivation rather than missplicing underlies the detrimental phenotype. To test whether heart failure could pharmacologically be reversed, we treated heterozygous Rbm20-R636Q knock-in (KI) mice with hesperadin, a potent CAMK2 inhibitor, which rescued both cardiac function and ventricular geometry. These data demonstrate that overactivation of CAMK2D underlies heart failure in RBM20 cardiomyopathy. Pharmacological inhibition of CAMK2D could therefore become the first cause-directed DCM therapy.

molecular biology↗

bioRxiv2024

SRSF2 transcriptional function maintains genome integrity during cell division

Recurrent mutations in serine / arginine-rich splicing factor 2 (SRSF2), particularly the proline-to-histidine substitution at position 95 (P95H), have been proposed to drive neoplastic diseases. SRSF2 plays pivotal roles in pre-mRNA processing and gene transcription. However, the precise impact of these diverse functions of SRSF2 on cancer cell behaviour remains unclear. Here, we show that deletion or homozygous P95H mutation of SRSF2 both cause extensive DNA damage leading to cell cycle arrest in vitro and in vivo, regardless of cell genotype. We mechanistically demonstrate that SRSF2 is required for efficient bi-directional transcription of DNA replication and repair genes, independent of its function in splicing. In contrast, SRSF2 haploinsufficiency induces DNA damage without halting the cell cycle in cancer cells. Inducing the heterozygous Srsf2 P95H mutation leads to clonal expansion of epidermal cells in mouse skin, but tumor formation is inhibited following exposure to carcinogens. To survive carcinogen treatment, cells containing the Srsf2 P95H mutation undergo substantial transcription rewiring that restores bi-directional gene expression. Thus, our study reveals crucial roles of SRSF2 beyond splicing and underscores its importance in regulating transcription to orchestrate the cell cycle along with the DNA damage response.

cell biology↗

bioRxiv2024

NMDtxDB: Data-driven identification and annotation of human NMD target transcripts

The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use general established rules. We introduce a dataset with 4 cell lines and combinations for SMG5, SMG6 and SMG7 knockdowns or SMG7 knockout. Based on this dataset, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information from Ensembl, Gencode consensus RiboSeq ORFs and OpenProt to enhance the CDS annotation of novel transcript isoforms. 302,889 transcripts were obtained from the transcriptome assembly process, out of which, 48,213 contain a premature stop codon and 6,433 are significantly up regulated in three or more comparisons of NMD active vs deficient cell lines. We present an in-depth view on these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.

bioinformatics↗

bioRxiv2024

Transverse Aortic COnstriction Multi-omics Analysis (TACOMA) uncovers pathophysiological cardiac molecular mechanisms

Time-course multi-omics data of a murine model of progressive heart failure induced by transverse aortic constriction (TAC) provide insights into the molecular mechanisms that are causatively involved in contractile failure and structural cardiac remodelling. We employ Illumina-based transcriptomics, Nanopore sequencing, and mass spectrometry-based proteomics on samples from the left ventricle (LV) and right ventricle (RV, RNA only) of the heart at 1, 7, 21, and 56 days following TAC and Sham surgery. Here, we present TACOMA, as an interactive web-application that integrates and visualizes transcriptomics and proteomics data collected in a TAC time-course experiment. TACOMA enables users to visualize the expression profile of known and novel genes and protein products thereof. Importantly, we capture alternative splicing events by assessing differential transcript and exon usage as well. Co-expression-based clustering algorithms and functional enrichment analysis revealed overrepresented annotations of biological processes and molecular functions at the protein and gene levels. To enhance data integration, TACOMA synchronizes transcriptomics and proteomics profiles, enabling cross-omics comparisons. With TACOMA (https://shiny.dieterichlab.org/app/tacoma), we offer a rich web-based resource to uncover molecular events and biological processes implicated in contractile failure and cardiac hypertrophy. For example, we highlight: (i) changes in metabolic genes and proteins in the time course of hypertrophic growth and contractile impairment; (ii) identification of RNA splicing changes in the expression of Tpm2 isoforms between RV and LV; and (iii) novel transcripts and genes likely contributing to the pathogenesis of heart failure. We plan to extend these data with additional environmental and genetic models of heart failure to decipher common and distinct molecular changes in heart diseases of different aetiologies. Database URLhttps://shiny.dieterichlab.org/app/tacoma

bioinformatics↗

bioRxiv2022

Magnetique: An interactive web application to explore transcriptome signatures of heart failure

Despite a recent increase in the number of RNA-seq datasets investigating heart failure (HF), accessibility and usability remain critical issues for medical researchers. We present Magnetique (https://shiny.dieterichlab.org/app/magnetique), an interactive web application to explore the transcriptional signatures of heart failure. We reanalyzed the Myocardial Applied Genomics Network RNA-seq dataset, one of the largest publicly available datasets of left ventricular RNA-seq samples from patients with dilated (DCM) or hypertrophic (HCM) cardiomyopathy, as well as unmatched non-failing hearts from organ donors and patient characteristics that allowed us to model confounding factors. Focusing on the DCM versus HCM contrast, we identified 201 differentially expressed genes and associated pathway signatures. Moreover, we predict underlying signaling networks based on inferred transcription factor activities. To the best of our knowledge, Magnetique is the first online application to provide an interactive view of the HF transcriptome by analyzing differential transcript isoform usage. Finally, another graphical view on statistically predicted RNA-binding protein to target transcript interactions complements the Magnetique web application. The source code for both the analyses (https://github.com/dieterich-lab/magnetiqueCode2022) and the web application (https://github.com/AnnekathrinSilvia/magnetique) is available to the public. We hope that our application will help users to uncover the molecular basis of heart failure.

bioinformatics↗

bioRxiv2021

Baltica: integrated splice junction usage analysis

Alternative splicing is a tightly regulated co- and post-transcriptional process contributing to the transcriptome diversity observed in eukaryotes. Several methods for detecting differential junction usage (DJU) from RNA sequencing (RNA-seq) datasets exist. Yet, efforts to integrate the results from DJU methods are lacking. Here, we present Baltica, a framework that provides workflows for quality control, de novo transcriptome assembly with StringTie2, and currently 4 DJU methods: rMATS, JunctionSeq, Majiq, and LeafCutter. Baltica puts the results from different DJU methods into context by integrating the results at the junction level. We present Baltica using 2 datasets, one containing known artificial transcripts (SIRVs) and the second dataset of paired Illumina and Oxford Nanopore Technologies RNA-seq. The data integration allows the user to compare the performance of the tools and reveals that JunctionSeq outperforms the other methods, in terms of F1 score, for both datasets. Finally, we demonstrate for the first time that meta-classifiers trained on scores of multiple methods outperform classifiers trained on scores of a single method, emphasizing the application of our data integration approach for differential splicing identification. Baltica is available at https://github.com/dieterich-lab/Baltica under MIT license.

bioinformatics↗

bioRxiv2020

Nonsense-mediated mRNA decay relies on "two-factor authentication" by SMG5-SMG7

Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that requires two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

molecular biology↗

bioRxiv2017

A study of the structural properties of sites modified by the O-linked 6-N-acetylglucosamine transferase

Protein O-GlcNAcylation (O-GlcNAc) is an essential post-translational modification (PTM) in higher eukaryotes. The O-linked {beta}-N-acetylglucosamine transferase (OGT), targets specific Serines and Threonines (S/T) in intracellular proteins. However, unlike phosphorylation, fewer than 25% of known O-GlcNAc sites match a clear sequence pattern. Accordingly, the three dimensional structures of O-GlcNAc sites were characterised to investigate the role of structure in molecular recognition. Of the 143/1,584 O-GlcNAc sites in 620 proteins were mapped to protein X-ray structures. The modified S/T were 1.7x more likely to be annotated in the REM465 field which defines missing residues in a protein structure, while 7 O-GlcNAc sites were solvent inaccessible and unlikely to be targeted by OGT. The 132/143 sites with complete backbone atoms clustered into 10 groups, but these were indistinguishable from clusters from unmodified S/T. This suggests there is no prevalent three-dimensional motif for OGT recognition. Predicted features from the 620 proteins were compared to unmodified S/T in O-GlcNAcylated proteins and globular proteins. The Jpred4 predicted secondary structure shows that modified S/T were more likely to be coils. 5/6 methods to predict intrinsic disorder indicated O-GlcNAcylated S/T to be significantly more disordered than unmodified S/T. Although the analysis did not find a pattern in the site three-dimensional structure, it revealed the residues around the modification site are likely to be disordered and suggests a potential role of secondary structure elements in OGT site recognition.

bioinformatics↗