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Pathogenic Proteotoxicity of Cryptic Splicing is Alleviated by Ubiquitination and ER-Phagy

ABSTRACT: RNA splicing and protein degradation systems allow the functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. Importantly, disruptive cryptic variants evade mRNA surveillance pathways and are translated into misfolded proteins. These spurious isoforms disrupt proteostasis causing cytosolic protein aggregates and ER stress. Proteotoxic exons activate unfolded protein response, causing CHOP-mediated cell death. In response to impaired splicing, eukaryotic cells enhance ubiquitination and ER-phagy to alleviate the pathogenic accumulation of proteotoxic isoforms. Our findings show how cryptic splicing-induced proteotoxicity can be mitigated, and provide insight into the molecular pathogenesis of spliceosome-associated diseases such as retinitis pigmentosa.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Retinal Pigment Epithelium, Cell Culture

SUBMITTER: Christian Münch

LAB HEAD: Dr. Christian Münch

PROVIDER: PXD050806 | Pride | 2024-09-25

REPOSITORIES: Pride

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			Action	DRS
	20240109_LU_LC2_MAA_DB_Cristian_Cell_F0.msf	Msf
	20240109_LU_LC2_MAA_DB_Cristian_Cell_F01.raw	Raw
	20240109_LU_LC2_MAA_DB_Cristian_Cell_F02.raw	Raw
	20240109_LU_LC2_MAA_DB_Cristian_Cell_F03.raw	Raw
	20240109_LU_LC2_MAA_DB_Cristian_Cell_F04.raw	Raw

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Pathogenic Proteotoxicity of Cryptic Splicing is Alleviated by Ubiquitination and ER-Phagy

Project description:RNA splicing and protein degradation systems allow the functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. Importantly, disruptive cryptic variants evade mRNA surveillance pathways and are translated into misfolded proteins. These spurious isoforms disrupt proteostasis causing cytosolic protein aggregates and ER stress. Proteotoxic exons activate unfolded protein response, causing CHOP-mediated cell death. In response to impaired splicing, eukaryotic cells enhance ubiquitination and ER-phagy to alleviate the pathogenic accumulation of proteotoxic isoforms. Our findings show how cryptic splicing-induced proteotoxicity can be mitigated, and provide insight into the molecular pathogenesis of spliceosome-associated diseases such as retinitis pigmentosa.

2024-09-25 | GSE272836 | GEO

Pathogenic Proteotoxicity of Cryptic Splicing is Alleviated by Ubiquitination and ER-Phagy

Project description:RNA splicing and protein degradation systems allow functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. Importantly, disruptive cryptic variants evade mRNA surveillance pathways and are translated into misfolded proteins. These spurious isoforms disrupt proteostasis causing proteotoxic aggregates, ER stress and CHOP-mediated cell death. In response to impaired splicing, eukaryotic cells enhance ubiquitination and ER-phagy to alleviate the pathogenic translation of proteotoxic exons. Our findings show how cryptic splicing-inducedproteotoxicity can be mitigated, and provide insight into the molecular pathogenesis of spliceosome-associated diseases, such as retinitis pigmentosa.

2024-09-25 | PXD051146 | Pride

The Pathogenic Impact of Cryptic Splicing is Alleviated by Ubiquitination and ER-Phagy

Project description:RNA splicing and the ubiquitin system allow the functional proteome to adapt in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here we show that the deregulation of the spliceosome B complex caused by USP39 deficiency leads to a novel splicing profile characterized by the use of cryptic 5′splice sites. Importantly, disruptive variants evade mRNA surveillance pathways and are translated into topologically incorrect proteins. These cryptic isoforms disrupt proteostasis and activate the unfolded protein response, causing ER stress-induced cell death. Human cells respond to this proteotoxicity by enhancing ubiquitin-mediated proteolysis and ER-phagy. Our findings show how cell death triggered by cryptic splicing can be mitigated, and provide insight into the molecular pathogenesis of diseases such as retinitis pigmentosa.

2024-09-26 | PXD041509 | Pride

Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing to prevent double-stranded RNA accumulation

Project description:Mitochondrial biogenesis relies on both the nuclear and the mitochondrial genomes, and the mechanisms that support their coordinated expression are not fully understood. Improper mitochondrial DNA expression can lead to inborn error of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic multi-substrate methyltransferase. We analyze genetic dependency, transcription, translation, and proteomic profiles of N6AMT1-depleted cells and report that N6AMT1 is necessary for the cytosolic translation of factors involved in mitochondrial RNA metabolism, including subunits of the mitochondrial RNase P. In the absence of N6AMT1, RNA processing and translation within mitochondria are impaired, while double-stranded RNA accumulates in mitochondrial RNA granules causing an interferon response. Our work highlights a cytosolic program required for proper mitochondrial biogenesis, with consequences on innate immunity.

2024-11-06 | PXD051180 | Pride

The E3 ligase TRIM25 impairs apoptotic cell death in colon carcinoma cells through hnRNPH1-triggered destabilization of caspase-7 mRNA

Project description:Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation, thereby contributing to apoptosis resistance of CRC cells towards chemotherapeutic drugs. We herein report the executioner caspase-7 being a further target of TRIM25. Results from gain and loss-of-function approaches and actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. Data from RNA pull-down assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA which is mediated by the PRY/SPRY domain which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein involved in the control of caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that hnRNPH1 and TRIM25 are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25 via hnRNPH1 implies a novel survival mechanism underlying chemotherapeutic drug resistance of CRC cells. Targeting of TRIM25 could therefore offer a promising strategy for reducing therapy resistance in CRC patients.

2023-01-05 | PXD037182 | Pride

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma

Project description:Resistance to proteasome inhibitors (PIs) is a ubiquitous clinical concern in multiple myeloma. We proposed that signaling-level responses after PI would reveal new means to enhance efficacy. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on spliceosome components. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis demonstrated broad-scale intron retention suggestive of PI-specific splicing interference. Direct spliceosome inhibition synergized with carfilzomib and showed potent anti-myeloma activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.

2020-02-21 | PXD012172 | Pride

Modeling of the U1 snRNP assembly pathway in alternative splicing in human cells using Petri nets

Project description:The investigation of spliceosomal processes is currently a topic of intense research in molecular biology. In the molecular mechanism of alternative splicing, a multi-protein–RNA complex – the spliceosome – plays a crucial role. To understand the biological processes of alternative splicing, it is essential to comprehend the biogenesis of the spliceosome. In this paper, we propose the first abstract model of the regulatory assembly pathway of the human spliceosomal subunit U1. Using Petri nets, we describe its highly ordered assembly that takes place in a stepwise manner.

2024-10-31 | MODEL2410300001 | BioModels

Identification of interacting proteins with U4 snRNA with and without USP39

2024-09-25 | PXD050688 | Pride

Complexome profiling of nuclear lysates from control and USP39-depleted HeLa cells

Project description:RNA splicing and protein degradation systems allow the functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. To explore the interactions of USP39 and the effect of its depletion in HeLa cells, we performed complexome profiling of nuclear lysates. We observed most USP39 stably integrated into the tri-snRNP complex and there is almost no free nuclear protein. More importantly, the relative abundance of tri-snRNP spliceosome complex was impaired in USP39-depleted cells. As previous reports indicated, USP39 is a regulator of tri-snRNP stability and its depletion decreased the levels of assembled U4/U6.U5 complexes.

2024-09-25 | PXD050738 | Pride

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Project description:Chemical splicing modulators that bind to the spliceosome have provided an attractive venue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. Yet, the biological effect of proteins containing translated intron sequences remains unclear. Here we identified a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) using genome-wide ribosome profiling and bio-orthogonal non-canonical amino-acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through JNK phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

2021-03-29 | GSE129305 | GEO

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