Biology subjects

Zavolan, M.

Publications and source records attributed to Zavolan, M..

At least 19 recordsLinked to original sources

bioRxiv2026

PAIP1 couples mRNA export to cytoplasmic mRNP remodeling and poly(A) homeostasis

Messenger ribonucleoproteins (mRNPs) acquire distinct protein compositions as they move from the nucleus to the cytoplasm, yet how this transition is coordinated and how inefficient remodeling affects downstream mRNA metabolism remains poorly understood. Here, we identify PAIP1 as a metazoan cofactor of the mRNA export ATPase DDX19. By binding a conserved N-terminal motif in DDX19, PAIP1 is recruited to the nuclear pore, where it promotes exchange of the nuclear poly(A)-binding protein PABPN1 for cytoplasmic PABPC1, thereby coupling mRNA export to cytoplasmic mRNP maturation. PAIP1 depletion alters cytoplasmic mRNP composition, mRNA stability and poly(A)-tail homeostasis. In Drosophila embryos, where poly(A)-tail regulation of maternal mRNAs directs early development, maternal PAIP1 depletion shortens poly(A)-tails, delays zygotic genome activation, and causes severe developmental defects. Our findings identify an export-coupled mRNP maturation pathway linking PABP exchange to downstream mRNA metabolism.

molecular biology↗

bioRxiv2026

SYBR Gold staining of single stranded nucleic acids is strongly influenced by the presence of guanosines

SYBR Gold is one of the most strongly fluorescent members of the SYBR family of asymmetrical cyanine dyes commonly used for staining double and single-stranded DNA and RNA. The dye fluorescence increases dramatically upon nucleic acid binding. The primary mode of binding to double-stranded DNA is through intercalation between the base-paired deoxyribonucleotides, while the precise mode of binding to single-stranded DNA and to RNA is less well understood. Here we show that the fluorescence activity of SYBR Gold is strongly influenced by the presence of guanines in single-stranded nucleic acids. We expect this insight to aid in the evaluation of SYBR Gold-based measurements of nucleic acid abundance and spark further studies into the binding behaviour of the SYBR family dyes.

molecular biology↗

bioRxiv2026

TORC1 spatial segregation and hysteresis are regulated by the small GTPase Arf1

TORC1 is a central regulator of growth, integrating inputs from kinases and signaling proteins. How TORC1 activity is regulated, is still not completely understood. The small GTPase Arf1 has been implicated in TORC1 regulation, but the underlying mechanism remains elusive. Here, we show that Arf1 controls TORC1 in an allele-specific manner under nutrient and heat stress in yeast. Unexpectedly, both the hyperactive mutant arf1-11 and the loss-of-function mutant arf1-18 reduce TORC1 activity, while other loss-of-function alleles did not. We reveal two distinct functions of Arf1: first, it ensures Golgi-to-vacuole/lysosome transport for the functional maintenance of these organelles; second, it regulates TORC1 activity at the ER-Golgi-vacuole interface. Hyperactive Arf1-11 drives sequestration of Kog1 and Tor1 into cytoplasmic foci, to which Arf1-11 can be equally recruited. Importantly, Arf1 also promotes TORC1 re-activation during stress recovery - so-called hysteresis. Our data reveal an unexpected dual function for Arf1 as a negative and a positive regulator of TORC1 activity in a context-dependent manner. TeaserThe small GPTase Arf1 acts as a dual switch for the key cellular growth regulator TORC1 by increasing or decreasing TORC1 activity depending on nutritional status and stress.

cell biology↗

bioRxiv2026

Histone H1 Variants Regulate Neurodevelopmental Transcriptional Programs in Autism with 16p11.2 deletion

BackgroundNeurodevelopmental disorders, including autism spectrum disorder, involve widespread transcriptional dysregulation. Copy number variations at 16p11.2 are among the strongest genetic risk factors for autism spectrum disorder, yet the molecular mechanisms by which these copy number variations contribute to neurodevelopmental pathology remain unclear. ResultsWe identify significant genetic associations between autism spectrum disorder susceptibility and the HIST1 histone gene cluster through genome-wide analysis. Transcriptomic profiling across post-mortem brain tissue, patient-derived neural progenitor cells, neurons, and cerebral organoids reveals consistent upregulation of linker histone variants H1.2 and H1.5 in idiopathic autism spectrum disorder and 16p11.2 hemi-deletion carriers, but not in schizophrenia or bipolar disorder. Functional assays demonstrate that dysregulated H1 expression disrupts gene networks involved in synaptic signaling, chromatin remodeling, and neural differentiation. Mechanistically, we link H1 upregulation to MAZ, a transcription factor encoded within the 16p11.2 locus. MAZ binds the promoter regions of H1 genes and represses their transcription. Knockdown of MAZ leads to H1 overexpression. H1 upregulation alone is sufficient to alter the expression of autism spectrum disorder-associated genes. ConclusionsOur findings define a MAZ-dependent regulation of H1 dosage as a critical chromatin-mediated mechanism contributing to transcriptional pathology in 16p11.2-associated autism spectrum disorder.

genomics↗

bioRxiv2026

Ribosome profiling reveals extensive translational alterations in steatohepatitis-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one the most lethal cancer types. While infections with hepatitis B or hepatitis C viruses remain the primary risk factors for HCC development worldwide, metabolic disease-associated steatohepatitis has become the fastest growing etiology of HCC, particularly in the West. Our study characterizes, for the first time, the remodeling of the translation landscape in steatohepatitis-associated HCC using ribosome profiling and mass spectrometry techniques. In striking contrast with the transcriptional alterations, which do not show strong dependence on associated co-morbidity, translational alterations are broader in the steatohepatitis compared to the viral background. These alterations affect hundreds of genes, whose products are often involved in subcellular protein localization. By quantifying the relationship between the ribosome occupancy of upstream or downstream open reading frames (uORFs or dORFs) and coding regions (CDSs), we provide evidence of uORFs attenuating the translation efficiency of CDSs in HCC. We also identify numerous novel translated regions, including from RNAs currently annotated as non-coding, some yielding peptides that can be reproducibly identified in mass spectrometry datasets. Our study thus provides novel molecular data on HCC with a focus on the steatohepatitis etiology, reveals unexpectedly extensive translational control in this co-morbidity, and gives multiple hints and candidate targets for follow-up experiments.

systems biology↗

bioRxiv2025

Heat shock induces silent ribosomes and reorganizes mRNA turnover

mRNAs associate with single or multiple ribosomes; these ribosomal assemblies -- monosomes and polysomes -- translate the mRNAs before degradation. The impact of heat stress on this mRNA turnover remains unclear. In heat-shocked yeast cells, the proportion of monosomes within the ribosomal assemblies rises without a corresponding increase in the number of mRNAs associated with them. As a result, most monosomes are devoid of mRNAs and silent, lacking translational initiation factors and proteins facilitating posttranslational folding. The accumulation of silent ribosomes generally reduces the rate of association of transcripts with the ribosomes. However, elevated temperatures enhance the ribosomal association of specific mRNAs, primarily those encoding heat-shock proteins, allowing them to balance their increased degradation rates. Additionally, reduced binding of the Xrn1 exonuclease to mRNAs diminishes the influence of codon optimality on mRNA stability. These mechanisms reorganize mRNA turnover to prioritize heat-shock protein synthesis over ribosome biogenesis.

genomics↗

bioRxiv2025

Regulation of mRNA polyadenylation governs mammalian body plan formation in gastruloids

The establishment of the body plan during gastrulation represents a hallmark of animal life. It emerges from the interplay of gene-regulatory programs and positional cues, yet how these signals are integrated post-transcriptionally remains largely unexplored. Here, we combine the scalability of mouse gastruloids with a single-cell CRISPR screening platform to functionally dissect germ layer specification at single-cell transcriptomic resolution. Focusing on post-transcriptional regulation, we systematically map drivers of mesodermal and endodermal fate and identify the deadenylase Cnot8. Loss of Cnot8 leads to widespread poly(A) tail elongation and transcript stabilization, shifting mesoderm differentiation toward ectopic notochord fate, thereby profoundly impacting axial patterning. Collectively, our findings identify mRNA deadenylation as a fundamental mechanism linking cellular identity with morphogenetic signaling during mammalian body plan formation.

developmental biology↗

bioRxiv2025

Identification of new interactors of eIF3f by endogenous proximity-dependent biotin labelling in human muscle cells.

Regulation of protein synthesis is central to maintaining skeletal muscle integrity and its understanding is important for the treatment of muscular and neuromuscular pathologies. The eIF3f subunit of the translation initiation factor eIF3 has a key role, as it stands at the crossroad between protein-synthesis-associated hypertrophy and MAFbx/atrogin-1-dependent. To decipher the molecular mechanisms underpinning the role of eIF3f in regulating muscle mass, we established a cellular model that enables interrogation of eIF3f functionality via identification of proximal interactors. Using CRISPR-Cas9 molecular scissors, we generated single cell clones of immortalised human muscle cells expressing eIF3f fused to the BirA biotin ligase (eIF3f-BioID1 chimera) from the endogenous EIF3F locus. Biotinylated proteins, representing interactors of eIF3f in nanometer range distance, were identified by streptavidin pull-downs and mass spectrometry. In both proliferating and differentiated muscle cells, the eIF3f-BioID1 chimera co-sedimented with ribosomal complexes in polysome profiles and interacted mainly with components of the eIF3 complex, and with the eIF4E, eIF4G, and eIF5 initiation factors. Surprisingly, we identified several nucleus-localised interactors of eIF3f, and the immunofluorescence analyses revealed a previously unknown nuclear localization of eIF3f in both myoblasts and myotubes. We also identified novel cytoplasmic partners of eIF3f, responsible for the maintenance of skeletal muscle ultrastructure (sarcomeric/Z-disc (SYNPO2) bound proteins) and proteins of the lysosomal compartment (LAMP1). The established tagging system should be useful to further advance studies of eIF3f function in hypertrophic and atrophic conditions in skeletal muscle.

molecular biology↗

bioRxiv2024

Current limitations in predicting mRNA translation with deep learning models

BackgroundThe design of nucleotide sequences with defined properties is long-standing problem in bioengineering. An important application is protein expression, be it in the context of research or the production of mRNA vaccines. The rate of protein synthesis depends on the 5 untranslated region (5UTR) of the mRNAs, and recently, deep learning models were proposed to predict the translation output of mRNAs from the 5UTR sequence. At the same time, large data sets of endogenous and reporter mRNA translation have become available. ResultsIn this study we use complementary data obtained in two different cell types to assess the accuracy and generality of currently available models of translation. We find that while performing well on the data sets on which they were trained, deep learning models do not generalize well to other data sets, in particular of endogenous mRNAs, which differ in many properties from reporter constructs. ConclusionsThese differences limit the ability of deep learning models to uncover mechanisms of translation control and to predict the impact of genetic variation. We suggest directions that combine high-throughput measurements and machine learning to unravel mechanisms of translation control and improve construct design.

bioinformatics↗

bioRxiv2023

CCAR1 promotes DNA repair via an unanticipated role in alternative splicing

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology directed repair (HDR) in the context of [~]18000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi Anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

molecular biology↗

bioRxiv2023

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

bioinformatics↗

bioRxiv2023

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding ofproteins with alpha helical domains

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we have quantified the expression of RPL39L in human pluripotent cells, cancer cell lines and tissue samples, and in mouse germ cells. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread proteasomal activity. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

molecular biology↗

bioRxiv2023

T helper cells exhibit a dynamic and reversible 3'UTR landscape.

3 untranslated regions (3UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBP) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localisation. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3UTR dynamics in T helper cells we investigated division-dependent alternative polyadenylation (APA). We generated 3 end UTR sequencing data from naive, activated, memory and regulatory CD4+ T cells. 3UTR length changes were estimated using a non-negative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNAseq dataset, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3UTRs. These results indicate that polyA site usage could play an important role in the control of cell fate decisions and homeostasis.

immunology↗

bioRxiv2022

Improved analysis of (e)CLIP data with RCRUNCH yields a compendium of RNA-binding protein binding sites and motifs

We present RCRUNCH, an end-to-end solution to CLIP data analysis for identification of binding sites and sequence specificity of RNA-binding proteins. RCRUNCH can analyze not only reads that map uniquely to the genome, but also those that map to multiple genome locations or across splice boundaries, and can consider various types of background in the estimation of read enrichment. By applying RCRUNCH to the eCLIP data from the ENCODE project, we have constructed a comprehensive and homogeneous resource of in vivo-bound RBP sequence motifs. RCRUNCH automates the reproducible analysis of CLIP data, enabling studies of post-transcriptional control of gene expression.

bioinformatics↗

bioRxiv2022

A novel approach to single cell analysis to reveal intrinsic differences in immune marker expression in unstimulated macrophages from BALB/c and C57BL/6 mouse strains

Macrophages are cells of the innate immune system that provide the first line of defense against pathogens. Their functional and morphological heterogeneity is well known, though the origin of this heterogeneity is still debated. Furthermore, while mouse strains differ in the type of immune responses that they mount to individual pathogens, the range of gene expression variation among their macrophages in the absence of a specific stimulus is not known. By applying single cell RNA sequencing we here reveal the gene expression variation in pre-stimulation macrophage populations from specific pathogen-free BALB/c and C57BL/6 mice, two mouse strains that give prototypical Th2 and Th1-biased immune responses, directed towards extracellular or intracellular pathogens, respectively. We show that intrinsic differences between the macrophages of these two strains are detectable before any specific stimulation and we place the gene expression profile of these cells within the range of variation that is measured upon in vitro stimulation with pro-inflammatory lipopolysaccharide (LPS) and interferon {gamma} (IFN), or anti-inflammatory IL-4. We find that C57BL/6 mice show stronger evidence of macrophage polarization than BALB/c mice, which could explain their resistance to pathogens such as Leishmania. Our computational methods for analyzing single cell RNA sequencing data, controlling for common sources of stochastic variation, can be more generally adopted to uncover biological variation between cell populations.

systems biology↗

bioRxiv2022

Frequent co-regulation of splicing and polyadenylation by RNA-binding proteins inferred with MAPP

Maturation of eukaryotic pre-mRNAs via splicing, 3 end cleavage and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although over 1500 proteins are associated with RNAs in human cells, their binding motifs, targets and functions still remain to be elucidated, especially in the complex environment of human tissues and in the context of diseases. To overcome the lack of methods for systematic and automated detection of sequence motif-guided changes in pre-mRNA processing based on RNA sequencing (RNA-seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). We demonstrate MAPPs functionality by applying it to RNA-seq data from 284 RBP knock-down experiments in the ENCODE project, from which MAPP not only infers position-dependent impact profiles of known regulators, but also reveals RBPs that modulate both the inclusion of cassette exons and the poly(A) site choice. Among these, the Polypyrimidine Tract Binding Protein 1 (PTBP1) has a similar activity in glioblastoma samples. This highlights the ability of MAPP to unveil global regulators of mRNA processing under physiological and pathological conditions.

systems biology↗

bioRxiv2021

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

physiology↗

bioRxiv2021

ZARP: An automated workflow for processing of RNA-seq data

RNA sequencing (RNA-seq) is a crucial technique for many scientific studies and multiple models, and software packages have been developed for the processing and analysis of such data. Given the plethora of available tools, choosing the most appropriate ones is a time-consuming process that requires an in-depth understanding of the data, as well as of the principles and parameters of each tool. In addition, packages designed for individual tasks are developed in different programming languages and have dependencies of various degrees of complexity, which renders their installation and execution challenging for users with limited computational expertise. The use of workflow languages and execution engines with support for virtualization and encapsulation options such as containers and Conda environments facilitates these tasks considerably. Computational workflows defined in those languages can be reliably shared with the scientific community, enhancing reusability, while improving reproducibility of results by making individual analysis steps more transparent. Here we present ZARP, a general purpose RNA-seq analysis workflow which builds on state-of-the-art software in the field to facilitate the analysis of RNA-seq data sets. ZARP is developed in the Snakemake workflow language using best software development practices. It can run locally or in a cluster environment, generating extensive reports not only of the data but also of the options utilized. It is built using modern technologies with the ultimate goal to reduce the hands-on time for bioinformaticians and non-expert users. ZARP is available under a permissive Open Source license and open to contributions by the scientific community. Contactmihaela.zavolan@unibas.ch, alexander.kanitz@unibas.ch

bioinformatics↗