Biology subjects

Gehring, N. H.

Publications and source records attributed to Gehring, N. H..

12 recordsLinked to original sources

bioRxiv2026

A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

Small cell lung cancer (SCLC) is among the deadliest cancers with excessive somatic alterations, expectedly resulting in immunogenic epitopes. However, patient benefit from immunotherapy is limited. We found abundant frameshift mutations in SCLC, regarded as highly immunogenic, counterbalanced by a hyperactive nonsense-mediated decay (NMD) pathway, responsible for frameshift-mRNA degradation. NMD activity correlated with tumor mutational burden (TMB) across cancers, suggesting that SCLC depends on NMD for cellular homeostasis and immune evasion. NMD inhibition impaired SCLC proliferation and induced ER stress-dependent apoptosis in a TMB-dependent manner, thereby enabling pharmacological inhibition in vivo which effectively controlled tumor growth. By integrating genome and transcriptome sequencing with MHC-I immunopeptidomics and functional in vitro and in vivo assays, we identified that NMD inhibition boosted neoantigen expression and presentation by tumor cells and increased T cell recognition, thus enhancing overall tumor immunogenicity and further improving immunotherapy efficacy in vivo. Our work shows that SCLC - as a TMBhigh cancer - relies on NMD for survival and immune escape, uncovering a novel TMB-dependent tractable vulnerability for this devastating disease.

cancer biology↗

bioRxiv2024

Functional implications of the interaction of the SARS-CoV-2 Nucleocapsid protein with factors involved in nonsense-mediated mRNA decay

The RNA genome of the SARS-CoV-2 virus encodes for four structural proteins, 16 non- structural proteins and nine putative accessory factors. A high throughput analysis of interactions between human and SARS-CoV-2 proteins identified multiple interactions of the structural Nucleocapsid (N) protein with RNA processing factors. The N-protein, which is responsible for packaging of the viral genomic RNA was found to interact with two RNA helicases, UPF1 and MOV10 that are involved in nonsense-mediated mRNA decay (NMD). Using a combination of biochemical and biophysical methods, we investigated the interaction of the SARS-CoV-2 N-protein with NMD factors at a molecular level. Our studies led us to identify the core NMD factor, UPF2, as an interactor of N. The viral N-protein engages UPF2 in multipartite interactions and can negate the stimulatory effect of UPF2 on UPF1 catalytic activity. N also inhibits UPF1 ATPase and unwinding activities by competing in binding to the RNA substrate. We further investigate the functional implications of inhibition of UPF1 catalytic activity by N in mammalian cells. The interplay of SARS-CoV-2 N with human UPF1 and UPF2 does not affect decay of host cell NMD targets but might play a role in stabilizing the viral RNA genome.

biochemistry↗

bioRxiv2024

SMG1:SMG8:SMG9-complex integrity maintains robustness of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. SMG1-mediated phosphorylation of the key NMD factor UPF1 is essential for NMD initiation and regulated by SMG9 and the C-terminus of SMG8. However, their specific roles in NMD regulation within intact cells remain partially understood. Here, we deleted the C-terminus of en-dogenous SMG8 in human cultured cells, which resulted in unchanged NMD activity. Cell lines lacking SMG8 and SMG9 showed slight NMD inhibition and unchanged UPF1 phosphorylation levels, but were sensitized to treatment with a SMG1 inhibitor (SMG1i). Transcriptome-wide analysis revealed the upregulation of NMD-annotated transcripts, which corresponded to synergistic effects of SMG1i concentration and SMG8 and SMG9 knock-out conditions. Moreover, the UPF1 interactome showed enrichment of various NMD factors in SMG8 or SMG9 knock-out cells and following SMG1i treatment, suggesting an accumulation of stalled NMD complexes at various stages of the NMD process. Together, our work uncovers important roles of SMG8 and SMG9 in maintaining NMD robustness in human cells.

molecular biology↗

bioRxiv2024

Rapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated transcriptome in human cells

The helicase UPF1 acts as the central essential factor in human nonsense-mediated mRNA decay (NMD) and is involved in various other mRNA degradation processes. Given its multifunctionality, distinguishing between mRNAs regulated directly and indirectly by UPF1 remains a critical challenge. We engineered two different conditional degron tags into endogenous UPF1 in human cell lines to probe the consequences of UPF1 rapid depletion. UPF1 degradation inhibits NMD within hours and strongly stabilizes endogenous NMD substrates, which can be classified into different groups based on their expression kinetics. Extended UPF1 depletion results in massive transcript and isoform alterations, partially driven by secondary effects. We define a high-confidence UPF1-regulated core set of transcripts, which consists mostly of NMD substrates. NMD-regulated genes are involved in brain development and the integrated stress response, among other biological processes. In summary, UPF1 degron systems rapidly inhibit NMD, providing valuable insights into its roles across various experimental systems.

molecular 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↗

bioRxiv2023

mRNA N1-2'O-methylation by CMTR1 affects NVL2 mRNA splicing

Cap0-mRNA is characterized by a 5-5triphosphate-linked N7-methylated guanosine(m7G). In higher eukaryotes, the methyltransferase CMTR1 additionally methylates the 2O-position of the penultimate mRNA nucleotide(N1) ribose (cap1-mRNA). While the m7G cap is essential for mRNA export and translation initiation by the eIF4F complex, the N1-2O-methylation prevents recognition of cap1-mRNA by the antiviral RNA receptors RIG-I and IFIT1, but a function beyond immunotolerance remained elusive. Here, we generated CMTR1-knockout(CMTR1-/-) cells and found that type-I-interferon(IFN-I) treatment resulted in IFIT1-mediated reduction of cell viability and broad mRNA translation. Consequently, stimulation of the antiviral receptor RIG-I in CMTR1-/- cells revealed an IFIT1 dependent dramatic reduction of IFN-I and chemokine protein induction, demonstrating the importance of N1-2O-methylation for antiviral responses. Additionally, IFN-I- and IFIT1-independent effects were observed: CMTR1-/- cells were smaller, divided slower, and exhibited a reduced transcription of mRNAs coding ribosomal proteins (RP), 5TOP-RNA and snoRNA host genes(SNHG). Additionally, proteome and transcriptome analysis revealed that expression of NVL2, an essential factor in ribosome biogenesis, is strongly suppressed by an alternative-splicing event of NVL2 mRNA in CMTR1-/- cells. This reduction could only be rescued by catalytically active CMTR1. Altogether, besides antiviral immunity N1-2O-methylation by CMTR1 has broad effects on cellular physiology and controls splicing of NVL2.

molecular biology↗

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↗

bioRxiv2021

Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing, mRNA export, mRNA translation, and nonsense-mediated mRNA decay (NMD) via various interacting peripheral proteins. The EJC-binding protein RNPS1 might serve two functions: it suppresses mis-splicing of cryptic splice sites and activates NMD in the cytoplasm. When analyzing the transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines, we find no evidence for RNPS1 being a globally essential NMD factor. However, various aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that about half of these RNPS1-dependent splicing events was fully or partially rescued by the expression of the isolated RRM domain of RNPS1, whereas other splicing events are regulated by its C-terminal domain. We identified many splicing-regulatory factors, including SR proteins and U1 snRNP components, that specifically interact with the C-terminus or with the RRM of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

molecular biology↗

bioRxiv2021

UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited only slightly impaired NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

molecular biology↗

bioRxiv2020

Single cell transcriptome sequencing on the Nanopore platform with ScNapBar

The current ecosystem of single cell RNA-seq platforms is rapidly expanding, but robust solutions for single cell and single molecule full-length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single cell sequencing method on the 10x Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g. for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing saturation is low. ScNapBar uses unique molecular identifier (UMI) or Naive Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore datasets. We further applied ScNapBar to pools of cells with an active or a silenced non-sense mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

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↗