Stefan Legewie’s research while affiliated with University of Stuttgart and other places

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Publications (159)


Coordinated alternative splicing decisions via stepwise exon definition
  • Preprint
  • File available

December 2024

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19 Reads

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Alternative splicing of pre-mRNA is a fundamental step in human gene regulation and aberrant slic-ing is tightly linked to diseases including cancer. Various splicing events, such as alternative exon (AE) choice and intron retention (IR), are controlled by a common molecular machinery, the spliceosome. However, it remains elusive how the regulation of spliceosome activity coordinately affects these splicing decisions. Here, we analyze a large-scale mutagenesis screen and genome-wide RNA sequencing data to show that IR and AE choice are tightly coupled, as IR products pri-marily accumulate in alternative exons showing intermediate inclusion levels. Using data-driven mathematical modeling, we reveal that multistep exon recognition by the spliceosome explains the observed AE-IR dependency for cis-acting sequence mutations and upon knockdown of trans-acting RNA-binding proteins. Furthermore, we show that multistep exon definition is frequently per-turbed in cancer cells, which leads to the coordinated deregulation of intron retention and AE choice. In conclusion, we showed that the spliceosome coordinates complex splicing decisions via stepwise exon definition, which potentially facilitates the search for common molecular mechanisms for mis-splicing in cancer.

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Pathogenic proteotoxicity of cryptic splicing is alleviated by ubiquitination and ER-phagy

November 2024

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167 Reads

Science

RNA splicing enables the functional adaptation of cells to changing contexts. Impaired splicing has been associated with diseases, including retinitis pigmentosa, but the underlying molecular mechanisms and cellular responses remain poorly understood. In this work, we report that deficiency of ubiquitin-specific protease 39 (USP39) in human cell lines, zebrafish larvae, and mice led to impaired spliceosome assembly and a cytotoxic splicing profile characterized by the use of cryptic 5′ splice sites. Disruptive cryptic variants evaded messenger RNA (mRNA) surveillance pathways and were translated into misfolded proteins, which caused proteotoxic aggregates, endoplasmic reticulum (ER) stress, and, ultimately, cell death. The detrimental consequence of splicing-induced proteotoxicity could be mitigated by up-regulating the ubiquitin-proteasome system and selective autophagy. Our findings provide insight into the molecular pathogenesis of spliceosome-associated diseases.


Transcriptional regulators ensuring specific gene expression and decision-making at high TGFβ doses

November 2024

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32 Reads

Life Science Alliance

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.


Zellen sind Individualisten — Entscheidungsprozesse auf Einzelzellebene

June 2024

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28 Reads

BIOspektrum

Decision-making is a fundamental aspect of life. However, our understanding of how cells encode and decode information to enable reliable fate decisions remains limited. Employing live cell imaging and automated analysis, our research unveils substantial heterogeneity in the cellular response to TGFβ and sheds light on the intricate link between the dynamics of SMAD signaling, the state of individual cells and their fate decisions.


CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

May 2024

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10 Reads

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. Motivation The CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing 1–8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS) 9–12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.


Figure 1
Figure 2
Figure 4: Evidence for feedforward regulation in SMAD-dependent gene expression
Figure 5: TFs shaping TGFβ target gene expression and cell fates
Transcriptional regulators ensuring specific gene expression and decision making at high TGFβ doses

April 2024

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43 Reads

TGFβ-signaling regulates cancer progression by controlling cell division, migration and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision making towards specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing and morphological assays to quantitatively link signaling, gene expression and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that the majority of TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision making.


Mechanisms generating network switches and their role in circadian clocks

March 2024

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44 Reads

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1 Citation

Journal of Biological Chemistry

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.


FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

July 2023

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70 Reads

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14 Citations

Molecular Cell

Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.


Modeling Cellular Signaling Variability Based on Single-Cell Data: The TGFβ-SMAD Signaling Pathway

April 2023

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26 Reads

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3 Citations

Methods in molecular biology (Clifton, N.J.)

Nongenetic heterogeneity is key to cellular decisions, as even genetically identical cells respond in very different ways to the same external stimulus, e.g., during cell differentiation or therapeutic treatment of disease. Strong heterogeneity is typically already observed at the level of signaling pathways that are the first sensors of external inputs and transmit information to the nucleus where decisions are made. Since heterogeneity arises from random fluctuations of cellular components, mathematical models are required to fully describe the phenomenon and to understand the dynamics of heterogeneous cell populations. Here, we review the experimental and theoretical literature on cellular signaling heterogeneity, with special focus on the TGFβ/SMAD signaling pathway.


State- and stimulus-specific dynamics of SMAD signaling determine fate decisions in individual cells

March 2023

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54 Reads

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7 Citations

Proceedings of the National Academy of Sciences

SMAD-mediated signaling regulates apoptosis, cell cycle arrest, and epithelial-to-mesenchymal transition to safeguard tissue homeostasis. However, it remains elusive how the relatively simple pathway can determine such a broad range of cell fate decisions and how it differentiates between varying ligands. Here, we systematically investigate how SMAD-mediated responses are modulated by various ligands of the transforming growth factor β (TGFβ) family and compare these ligand responses in quiescent and proliferating MCF10A cells. We find that the nature of the phenotypic response is mainly determined by the proliferation status, with migration and cell cycle arrest being dominant in proliferating cells for all tested TGFβ family ligands, whereas cell death is the major outcome in quiescent cells. In both quiescent and proliferating cells, the identity of the ligand modulates the strength of the phenotypic response proportional to the dynamics of induced SMAD nuclear-to-cytoplasmic translocation and, as a consequence, the corresponding gene expression changes. Interestingly, the proliferation state of a cell has little impact on the set of genes induced by SMAD signaling; instead, it modulates the relative cellular sensitivity to TGFβ superfamily members. Taken together, diversity of SMAD-mediated responses is mediated by differing cellular states, which determine ligand sensitivity and phenotypic effects, while the pathway itself merely serves as a quantitative relay from the cell membrane to the nucleus.


Citations (22)


... Several RNAbinding proteins (RBPs), such as PTBP1, hnRNPLL, and XAB2, have been identified as coregulators in various sets of intron retention [48][49][50]. More recently, it has been reported that far upstream bingding protein 1 (FUBP1) function as a general splicing at 3' splice site, with a crucial role in promoting efficient splicing of long intron [51]. Our mechanistic analysis revealed that KHSRP modulates pre-mRNA splicing, especially intron retention. ...

Reference:

KHSRP ameliorates acute liver failure by regulating pre-mRNA splicing through its interaction with SF3B1
FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns
  • Citing Article
  • July 2023

Molecular Cell

... Current evidence suggests that the temporal dynamics of SMAD signaling play an important role in cellular decision-making. Using time-lapse microscopy of individual MCF10A cells, we found that transient SMAD2 dynamics are associated with increased cell motility, whereas sustained SMAD2 dynamics, in addition, induce cell cycle arrest and further reinforced cellular motility toward EMT (Strasen et al, 2018;Bohn et al, 2023). Comparable results were observed in pancreatic cancer cell lines, where a sustained SMAD signal was required for cell cycle arrest (Nicolás & Hill, 2003). ...

State- and stimulus-specific dynamics of SMAD signaling determine fate decisions in individual cells
  • Citing Article
  • March 2023

Proceedings of the National Academy of Sciences

... At molecular level, this process is guaranteed by an important macromolecular complex, the spliceosome, and a combination of several proteins, which cooperate to regulate a series of RNA-RNA and RNA-protein interactions (Tao et al., 2024). In general, by binding regulatory sequence elements of pre-mRNAs, hnRNP can act as either an activator or repressor of spliceosome recruitment, promoting or suppressing the inclusion of an exon, thus contributing to the generation of different mRNA transcripts (Horn et al., 2023). hnRNPs-preRNA binding can occur in both intronic and exonic domains, and influences the regulatory mechanisms of mRNA and its fate in the cytoplasm (Garcia-Maurino et al., 2017) ( Figure 1). ...

Position-dependent effects of RNA-binding proteins in the context of co-transcriptional splicing

npj Systems Biology and Applications

... AS also affects cell death, with isoforms like MCL-1 S and BCL-xS promoting apoptosis [51,52]. Additionally, splicing alters immune responses, as shown by CD19 exon 2 skipping, which impairs CAR-T therapy in leukemia [53]. Drug resistance is driven by isoforms like FGFR3-S and RAD51, which confer cancer therapy resistance [54,55]. ...

High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance

... For downstream kinase activities, a spatial stochastic model examined the opposing roles of progesterone receptor (PR) phosphorylation mediated by ERK kinase [180]. An SDE model was also utilized to describe the temporal stochastic bursting of the TGF-β/SMAD signaling pathway in single cells [181]. Stochastic fluctuations were introduced in the reactions at the receptor level, as receptor dynamics are rate-limiting in TGF-β signaling [182]. ...

Data-based stochastic modeling reveals sources of activity bursts in single-cell TGF-β signaling

... We input the DNA sequence and five half-life time features to scXpresso. However, certain transcript features, which are related to the half-life time features, can predict zeros in the scRNA-seq data (Lipnitskaya et al., 2022). Whether the observed zeros in scRNA-seq data are technical artifacts or biologically informative is an ongoing debate. ...

Machine learning-assisted identification of factors contributing to the technical variability between bulk and single-cell RNA-seq experiments

... Based on the results, all SNPs found in F2-crossed local chickens were located in the exon region. An exon is a part of DNA that remains during mRNA maturation [31]. Mutations in exons can affect gene expression because this region includes a coding sequence, the area that is translated into amino acids. ...

Exon Definition Facilitates Reliable Control of Alternative Splicing in the RON Proto-Oncogene
  • Citing Article
  • March 2020

Biophysical Journal

... Discrepancies observed between transcription and translation levels in light-treated cells imply a significant role in posttranscriptional regulation. Differences in protein and mRNA degradation rates, as well as translational and post-translational modifications in protein processing, influence the relationship between mRNA levels and protein expression (Becker et al. 2018;Du et al. 2021;Yang et al. 2020). Consequently, the integration of transcriptomic and proteomic analyses is essential for a comprehensive understanding of the molecular mechanisms underlying blue light irradiation in E. coli. ...

Quantifying post-transcriptional regulation in the development of Drosophila melanogaster

... Indeed, over one third of the splicing events in humans show complex splicing patterns combining multiple AS decisions at the same splice junction (24,25). Large-scale mutagenesis experiments showed that even a three-exon minigene can produce multiple isoforms by combining different splicing decisions already in WT and that mutations may activate cryptic splice sites further enriching the splicing choices (26,27). Thus, a single metric, such as PSI (percent spliced-in) for AE, is not sufficient to quantify the complex splicing decisions. ...

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

... We also investigated mutations affecting U2AF2, a key pre-mRNA splicing factor. 32 The mutation most strongly predicted to disrupt U2AF2 binding affinity has been confirmed to be associated with ataxia-oculomotor apraxia syndrome (rs1563963464), and EMSA confirmed its impact on U2AF2-RNA interactions ( Figures 6E and 6F). These findings offer valuable insights into the molecular mechanisms by which these mutations contribute to disease. ...

In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors

Genome Research