Biology subjects

Dieterich, C.

Publications and source records attributed to Dieterich, C..

At least 19 recordsLinked to original sources

bioRxiv2026

WarpDemuX-custom - Multiplexed sequence-specific direct RNA sequencing

Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA and modifications at single-molecule resolution. Although originally developed for polyadenylated RNAs, protocol adaptations have extended its application to non-polyadenylated RNA species, including tRNAs and rRNAs. However, the lack of open and flexible (de)multiplexing solutions remains a limitation for large-scale sequencing studies. Existing community-driven approaches enable multiplexing of polyadenylated RNAs and, more recently, tRNAs, but do not fully address the challenges of targeted sequence-specific Nanopore sequencing of non-polyadenylated RNAs. Here, we present an improved sequence-specific direct RNA sequencing protocol for ribosomal RNAs, achieving enhanced capture and longer read lengths as demonstrated for human 28S rRNA. We complement this protocol with an expanded WarpDemuX software toolbox for flexible sequence-specific (de)multiplexing. We validate the approach using human cell lines carrying knockouts of different C/D box snoRNAs involved in site-specific 2'-O-ribose methylation of 28S rRNA. Our results demonstrate efficient multiplexed detection of sequence-specific RNA molecules and associated modification changes. While established here using rRNA, we validate our approach on another panel of non-polyadenylated RNA species, providing an open and scalable framework for targeted direct RNA sequencing and RNA modification analysis

bioinformatics↗

bioRxiv2026

Eukaryotic tRNA ligases mediate RNA break repair

RNA is continuously exposed to damage during physiological metabolism and stress, yet cellular responses to RNA damage remain less understood than DNA repair pathways. RNA strand breaks are particularly deleterious because they generate chemically incompatible RNA ends. Eukaryotic tRNA ligases have been implicated in RNA processing and repair, but whether they function as general RNA repair enzymes remains unresolved. Here, we show that the evolutionarily divergent tRNA ligases, human RTCB and fungal Trl1, mediate RNA break repair (RBR) targeting ribosomes and other ribonucleoprotein (RNP) complexes. Using direct RNA nanopore sequencing, we map these repair events at nucleotide resolution, demonstrating that tRNA ligases repair breaks in ribosomal RNA and restore translational activity of repaired ribosomes. We further identify repair across additional structured cellular RNAs. We show that loss of RBR activity leads to RNA fragmentation in human cells and impairs cell viability upon oxidative stress. Together, these findings uncover a broader role for eukaryotic tRNA ligases in repairing RNA breaks and maintaining transcriptome integrity.

biochemistry↗

bioRxiv2026

Muscle LIM Protein is a Non-Canonical RNA-Binding Protein that Engages in RNA-Dependent Cytoskeletal Assemblies

Muscle LIM Protein (MLP) is a key component of the cardiomyocyte Z disc that is essential for sarcomere integrity and cardiac homeostasis. Accordingly, loss of MLP function results in (cardio)myopathy phenotypes in animal models and human patients. In this study, we present the first RNA-binding proteome (RBPome) of human cardiomyocytes using enhanced RNA interactome capture (eRIC). Data integration with existing RBPome datasets identified MLP as an evolutionary conserved and previously unrecognized non-canonical RNA-binding protein (RBP) in cardiomyocytes. Using complementary biochemical approaches, we confirmed direct RNA association of MLP. Moreover, RNA integrity was required for MLP interactions with cytoskeletal proteins and for the formation of higher-order MLP-containing assemblies, revealed by co-immunoprecipitation and RNA-dependent sedimentation profiling. Subcellular fractionation and imaging analyses indicated that MLPs association with cytoskeletal complexes may be weaker and more dynamic compared to core sarcomere proteins like -actinin. Domain mapping identified two glycine-rich regions within MLP as RNA contact sites, and RNA-binding-deficient MLP mutants showed impaired association with cytoskeletal complexes. Together, our findings suggest RNA binding as a regulated property of MLP and uncover an RNA-dependent layer of cytoskeletal organization in cardiomyocytes.

molecular biology↗

bioRxiv2026

CAPRINI-M: An AI-curated Cardiac-Specific Atlas of Protein Interactions in Mice

MotivationProtein-protein interactions are fundamental to cardiovascular disease biology, but the corresponding knowledge is dispersed across the literature and heterogeneous databases, making systematic curation time-consuming. Moreover, many existing PPI resources may be biased and lack detailed information on structural interaction interfaces or associated thermodynamic parameters. ResultsWe present CAPRINI-M (CArdiac PRotein INteractions In Mice), a web-based tool hosting an AI-curated atlas of cardiac protein interactions. We mined 9,105 cardiobiology manuscripts and used open-source LLMs (LLaMA-3.3 70B) to extract 11,189 protein-protein interactions. We then used AlphaFold3 to infer interaction interfaces, estimate thermodynamic properties related to complex stability, and predict the likelihood that each protein pair forms a complex. In our benchmarking analysis, CAPRINI-M showed stronger performance than the comparator PPI resources tested here. Predicted interaction favourability also agreed with published experimental evidence, with lower predicted Gibbs free energy associated with experimentally preferred binding partners. Overall, CAPRINI-M provides a more comprehensive, mechanistically annotated view of cardiovascular disease-relevant protein-protein interactions by integrating literature evidence with structural, interface-level, and stability-related information. AvailabilityThe CAPRINI-M web application is available at https://shiny.dieterichlab.org/app/caprinim. The source code used in this study is linked in the manuscripts Availability section.

bioinformatics↗

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

QutRNA2: Robust tRNA modification discovery from Nanopore direct tRNA sequencing

Transfer RNAs (tRNAs) are essential for protein synthesis and are extensively modified to ensure their structure and function. Direct RNA sequencing with Oxford Nanopore Technologies enables positional modification analysis but is challenged by tRNAs short length, redundancy, and dense modifications. We present QutRNA2, a scalable workflow featuring GPU-accelerated local alignment, statistical filtering, pairwise error profile comparison, and customizable visualization. Achieving up to 25-fold speed gains over CPU methods, QutRNA2 identifies enzyme-dependent modifications in nuclear- and mitochondrial-encoded tRNAs, demonstrated in human and mouse samples. This open-source solution provides a comprehensive, multiplexing-compatible framework for tRNA analysis, addressing a key gap in current tools.

bioinformatics↗

bioRxiv2025

Reading papers: Extraction of molecular interaction networks with large language models

MotivationSignalling occurs within and across cells and orchestrates essential cellular processes in complex tissues. Cell signalling involves several different components, including protein-protein interactions (PPI) and transcription factors (TF), to promoter binding in gene regulatory networks (GRNs). Dynamically changing conditions oftentimes lead to the rewiring of cellular communication networks. Computational modelling approaches typically rely on databases of possible molecular interactions. Evidently, manual curation of databases is time-consuming and automatic relation extraction from scientific literature would greatly support our strive to understand molecular mechanisms. To ease this process, we reason that prompt-based data mining with Large Language Models (LLMs) could be used to extract information from relevant scientific publications. ApproachIn our work, we use open-source LLMs to mine an annotated corpus of molecular interactions. We focus on the extraction of entity relations between proteins, as exemplified in protein-protein interaction networks, and transcription factor to target gene relations, as exemplified in gene regulatory networks. ResultsWe obtain promising evaluation results as measured by precision, recall and F1-score for the extraction of PPI relations: 87%, 70% and 71% and 77%, 57% and 62% for GRN relation extraction over a large corpus of short (average 331 tokens) scientific texts. AvailabilityCodes with scripts and results have been provided in: https://github.com/dieterich-lab/LLM_Relations.

bioinformatics↗

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↗

bioRxiv2025

WarpDemuX-tRNA: barcode multiplexing for nanopore tRNA sequencing

Transfer RNA (tRNA) plays an essential role in protein translation and tRNA modifications are important to their function. Recently, nanopore direct RNA sequencing (dRNA-seq) has shown promising results in the detection of complex tRNA modifications. However, its wider adoption in the tRNA field has been limited by a lack of (de)multiplexing solutions. Here, we present WarpDemuX-tRNA: an extension to the WarpDemuX method specifically optimized for multiplexed nanopore tRNA sequencing. Using consensus-based signal analysis using (soft) Dynamic Time Warping and barycenter averaging, our approach improves barcode feature generation and achieves more robust barcode identification. WarpDemuX-tRNA outperforms the original method and achieves 99% precision and 95% recovery for four barcodes, while reducing computational complexity and runtime to six minutes per one million reads. WarpDemuX-tRNA is an open-source and free-to-use solution to high-throughput nanopore tRNA sequencing, facilitating more accessible, cost-effective and high-throughput studies of tRNA modifications and their regulatory mechanisms.

bioinformatics↗

bioRxiv2025

The two-step purification method ViREn identifies a single NSUN6-mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover

Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes including RNA stability and translation. Although advances in analytical methods have improved detection sensitivity, the precise mapping of RNA modifications at single-base resolution remains challenging. Especially for low abundant viral RNAs extracted from infected cells, requirements for sensitivity and purity limit accuracy and reproducibility. Here we report the two-step method ViREn for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a high-confidence 5-methylcytosine (m5C) site in DENV gRNA, orthogonally validated by Illumina-based bisulfite sequencing and direct RNA sequencing by Nanopore Oxford Technologies. Strikingly, this m5C modification was exclusively detected in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on low abundance viral gRNA with unprecedented precision and sensitivity and facilitates mechanistic studies into the role of RNA modification in virus replication.

biochemistry↗

bioRxiv2025

MoDorado: Enhanced detection of tRNA modifications in nanopore sequencing by off-label use of modification callers

Rapid and accurate identification of tRNA modifications is crucial for understanding their role in protein translation and disease. However, their detection on tRNAs is challenging due to their high modification density. Recently, modification calling models for nanopore direct RNA sequencing became available for pseudouridine ({Psi}), m6A, inosine and m5C, as part of the Dorado basecaller. Applying the {Psi} model to tRNAs, we have mapped both known and novel {Psi} sites in Schizosaccharomyces pombe and assigned the responsible pseudouridine synthetases. Furthermore, we have developed MoDorado, an algorithm to detect modifications beyond those used in model training ("off-label use") by measuring prediction differences of pre-trained machine learning models. By leveraging the {Psi}/m6A/inosine/m5C models, MoDorado detected seven additional modifications (ncm5U, mcm5U, mcm5s2U, m7G, queuosine, m1A, and i6A), thus generating a tRNA modification map of S. pombe. This work demonstrates the potential of pre-trained models in determining the intricate landscape of tRNA modifications.

molecular biology↗

bioRxiv2025

Extending scope and power of circular RNA researchwith circtools 2.0

Circular RNAs (circRNAs) play a pivotal role in gene regulation, acting as transcriptional modulators at various cellular levels and exhibit remarkable specificity across cell types and developmental stages. Detecting circRNAs from high-throughput RNA-seq data presents significant challenges, as it requires specialized tools to identify back-splice junctions (BSJ) in raw sequencing data. To address this need, we previously developed circtools, a robust and user-friendly software for circRNA detection, downstream analysis, and wet-lab integration. Building on this foundation, we now introduce circtools 2.0, a significantly enhanced framework designed to advance computational and support experimental analysis for circular RNA research. Novel key features of circtools 2.0 include: (1) an ensemble approach to circRNA detection with improved recall rates, (2) a module for automated design of circRNA padlock probes for the Xenium spatial trancriptomics platform, (3) circRNA conservation analysis across animal model species, and (4) support for the Oxford Nanopore sequencing platform for full-length circRNA detection. Availabilitycirctools 2.0 is available at github.com/jakobilab/circtools and licensed under GPLv3.0. Contacttjakobi@arizona.edu

bioinformatics↗

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

A functional screen uncovers circular RNAs regulating excitatory synaptogenesis in hippocampal neurons

Circular RNAs (circRNAs) are an expanding class of largely unexplored RNAs which are prominently enriched in the mammalian brain. Here, we systematically interrogated their role in excitatory synaptogenesis of rat hippocampal neurons using RNA interference. Thereby, we identified seven circRNAs as negative regulators of excitatory synapse formation, many of which contain high-affinity microRNA binding sites. Knockdown of one of these candidates, circRERE, surprisingly promoted the formation of electrophysiologically silent synapses. Mechanistically, circRERE knockdown resulted in a preferential upregulation of synaptic mRNAs containing binding sites for miR-128-3p because of a reduced protective interaction between miR-128-3p and circRERE. Accordingly, overexpression of circRERE rescued exaggerated synapse formation upon circRERE knockdown in a miR-128-3p binding site-specific manner. Overall, our results uncover circRERE-mediated stabilization of miR-128-3p as a novel mechanism to restrict the formation of silent excitatory synaptic co-clusters and more generally implicate circRNA-dependent microRNA regulation in the control of synapse development and function.

neuroscience↗

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↗

bioRxiv2023

mAFiA: Detecting m6A at single-molecular resolution via direct-RNA sequencing

Direct-RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. Here, we report on the synthesis of such samples and the development of a bespoke algorithm that accurately detects single m6A nucleotides on single molecules in both synthetic RNAs and natural mRNA.

bioinformatics↗

bioRxiv2022

Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of DCM patients harbor heritable mutations which are amenable to CRISPR-based gene therapy1. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart2. We employed a combination of the viral gene transfer vector AAVMYO with superior targeting specificity of heart muscle tissue3 and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive and arrhythmogenic DCM4. Using optimized conditions, we could improve splice defects in human iPSC-derived cardiomyocytes (iPSC-CMs) and repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restored the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reached wildtype levels. Single-nuclei RNA sequencing (snRNA-seq) uncovered restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing (WGS) revealed no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

genetics↗