Biology subjects

Stoecklin, G.

Publications and source records attributed to Stoecklin, G..

11 recordsLinked to original sources

bioRxiv2026

Alternative polyadenylation mediated by CPSF5 regulates pro-inflammatory gene expression

The majority of mammalian genes contains more than one functional cleavage and polyadenylation site, so that selection of alternative sites leads to different 3 ends of the mature transcripts. This mechanism can lead to the exclusion or retention of regulatory sequence elements, which affects post-transcriptional regulation of gene expression, such as RNA stability, localization or translation efficiency. We used 3 end sequencing to assess alternative polyadenylation during the response of macrophages to LPS, and observed a strong global shift towards proximal polyadenylation sites. This was accompanied by a decreased expression of cleavage and polyadenylation factors, including CPSF5, which is known to favor selection of distal polyadenylation sites. Upon depletion of CPSF5 in macrophage, we observed global transcript shortening, and an induction of TNF and other pro-inflammatory cytokines without LPS-stimulation. Analysis of RNA-seq data from monocytes of sepsis patients revealed that CPSF5 expression and alternative polyadenylation are also affected in vivo. Usage of distal polyadenylation sites showed a negative correlation with TNF mRNA expression in human monocytes. Our data suggest that transcript shortening mediated by CPSF5 repression contributes to the induction of pro-inflammatory genes.

molecular biology↗

bioRxiv2026

Loss of cardiomyocyte eukaryotic elongation factor 1 A2 in adult mice triggers cardiomyopathy due to defective proteostasis

Eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to ribosomes but also has additional, non-canonical functions. Mammals express two paralogs: eEF1A1 is ubiquitous, whereas eEF1A2 is confined to adult cardiomyocytes, skeletal myocytes, and neurons. Mutations in EEF1A2 cause cardiomyopathy, but underlying mechanisms remain unclear. Using adult, cardiomyocyte-specific Eef1a2 knock-out (Eef1a2-cKO) and Eef1a1/Eef1a2 double knock-out mice, we show that Eef1a2-cKO animals develop cardiomyopathy with increased mortality, systolic dysfunction, and fibrosis, despite unchanged global protein synthesis, while double knock-out mice die early in a sudden manner. Multi-omics analyses reveal post-transcriptional upregulation of ribosomal proteins and translational regulators in both models. Eef1a2-cKO hearts accumulate autophagosomes and protein aggregates, indicating defective autophagy. Mechanistically, we found that eEF1A2 functions as a chaperone supporting protein folding and proteostasis in cardiomyocytes. Early Rapamycin treatment (mTORC1 inhibition) normalizes systolic heart function and survival in Eef1a2-cKO mice and clears autophagosomes and protein aggregates. Thus, eEF1A2 maintains cardiac proteostasis, and mTORC1 inhibition may represent a therapeutic strategy for patients with EEF1A2 mutations.

physiology↗

bioRxiv2026

Comprehensive analysis of nonsense-mediated mRNA decay targets and activity in cardiomyocytes

Nonsense-mediated mRNA decay (NMD) serves as a mechanism to suppress the expression of mutant alleles containing premature termination codons, limit the expression of aberrantly spliced transcript isoforms, and control the expression of numerous regular genes. While the principles by which NMD recognizes target transcripts are well understood, much less is known about the range of mRNAs subject to NMD in tissues and specialized cell types. Here we describe the landscape of genes whose expression is controlled by NMD in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CM), using small read RNA sequencing in combination with a potent inhibitor of SMG1, a kinase essential for NMD. We find that NMD targets are highly conserved between iPSC-CM lines derived from two healthy individuals. Beyond gene level analysis, we identify individual exon and intron RNA sequences that strongly accumulate upon SMG1 inhibition. Using the cardiac NMD targets identified at gene, exon and intron level, we then demonstrate reduced NMD efficiency upon knockdown of two essential NMD factors, UPF1 and UPF2, in iPSC-CM. Our analysis demonstrates that quantifying the transcriptome-wide response to SMG1 inhibition represents a highly sensitive approach to assess global activity of the NMD pathway.

molecular biology↗

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

MEK inhibition induces AXIN1 loss in colorectal cancer by mTOR associated suppression of protein synthesis

AXIN1 is a central regulatory hub of many oncogenic pathways in colorectal cancer (CRC). As the main scaffold protein and least abundant component of the beta-catenin destruction complex, changes in AXIN1 levels affect Wnt signaling output. We show that targeting the Ras-MAPK pathway by MEK1/2 inhibitors induces AXIN1 loss across a panel of CRC cell lines and patient-derived organoids. GSK3B inhibition similarly reduced AXIN1 levels, yet by distinct mechanisms. MEK1/2 causes a reduction of AXIN1 transcript levels, but neither affects protein stability nor post-translational modifications of AXIN1. In contrast, GSK3B inhibition induces rapid AXIN1 degradation. Prevention of AXIN1 loss by co-treatment with tankyrase inhibitors was much stronger for GSK3B than for MEK1/2 inhibition. Using isogenic CRC cell lines and murine intestinal organoids, we show that APC truncations strongly reduce basal AXIN1 levels, but do not alter dynamics of AXIN1 loss upon MEK1/2 inhibition. Polysome profiling and Ribo-Seq revealed that MEK1/2 inhibition reduces global protein synthesis via an mTOR dependent pathway. This translational repression is sufficient to cause significant AXIN1 loss, as treatment with mTOR inhibitors phenocopies the effect of MEK1/2 inhibitors. Our study demonstrates that AXIN1 protein homeostasis is critically controlled by Ras-MAPK signaling at the level of protein synthesis, and that MEK1/2 inhibitors cause AXIN1 loss by translational repression.

cancer biology↗

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

RNA modifications on Adenosine co-Regulate Macrophage Function

Macrophages are a highly plastic innate immune cell subset that depends on environmental cues to activate, execute and resolve inflammatory responses. This plasticity of function is mirrored by the diversity of RNA modifications that dynamically decorate macrophage transcripts. Here, using the mouse macrophage line RAW 264.7 (RAW), we addressed the combinatorial effect of two major mRNA modifications: adenosine to inosine (A-to-I) deamination by ADAR1 and adenosine N6-methylation (m6A) by METTL3. Using both short-read and single molecule sequencing on RAW macrophages with genetic deletions of ADAR1 or METTL3, we identified transcripts that were modified by both enzymes, with specific functional outcomes on macrophage activation. While m6A levels remained relatively stable even in the absence of ADAR1, loss of METTL3 led to a global reduction in A-to-I editing levels. This interrelation was most apparent when m6A sites were distant from sites of deamination, suggesting a possible function of m6A in ADAR1-mediated editing. Using a dual reporter cell line where guided ADAR1 recruitment can be measured via eGFP reactivation, we observed that m6A modification of ADAR-engager guide RNAs substantially improved targeted RNA editing. Overall, we report the first example of an interdependence between modifications, which can also be therapeutically exploited.

immunology↗

bioRxiv2023

GADD34 mRNA turnover controls the molecular memory of cellular stress experience

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of Growth Arrest and DNA Damage inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We established that the 3 untranslated region of PPP1R15A mRNA contains an active AU-rich element recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a key step in establishing the ISR molecular memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

cell biology↗

bioRxiv2022

Temporal control of the integrated stress response by a stochastic molecular switch

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

cell biology↗

bioRxiv2019

CDK1 couples proliferation with protein synthesis

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extra-mitotic function of CDK1. Dephosphorylation of eIF2 and S6K1 signaling were found to act downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5TOP mRNAs, which includes mRNAs encoding for ribosomal proteins and several translation factors. This effect requires the 5TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Taken together, our results show that CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

biochemistry↗

bioRxiv2018

DNA and RNA binding mediate the toxicity of arginine-rich peptides encoded by C9ORF72 GGGGCC repeats

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

neuroscience↗