Pub Date : 2025-11-20DOI: 10.1016/j.molcel.2025.10.028
Stephanie Patchett, Seyed Arad Moghadasi, Ankita Shukla, Farid El Oualid, Beatrix M. Ueberheide, Shaun K. Olsen, Tony T. Huang
In eukaryotes, each ribosomal subunit includes a ribosomal protein (RP) that is encoded as a fusion protein with ubiquitin (Ub). In yeast, each Ub-RP fusion requires processing by deubiquitylating enzymes (DUBs) to generate ribosome assembly-competent RPs and contribute to the cellular Ub pool. However, how Ub-RP fusions are processed by DUBs in human cells remains unclear. Here, we discovered that Ub-RPs are substrates of the Ub-fusion degradation (UFD) pathway in human cells via lysine 29 and 48 (K29/K48)-specific ubiquitylation and proteasomal degradation. We identified a pool of DUBs that catalytically process Ub-RPs, as well as DUBs that physically occlude Ub-RP interaction with UFD pathway Ub E3 ligases to prevent their degradation in a non-catalytic manner. Our results suggest that DUBs both process and stabilize Ub-RPs, whereas the UFD pathway regulates levels of Ub-RPs that cannot be fully processed by DUBs to fine-tune protein homeostasis.
{"title":"Deubiquitinases cleave ubiquitin-fused ribosomal proteins and physically counteract their targeting to the UFD pathway","authors":"Stephanie Patchett, Seyed Arad Moghadasi, Ankita Shukla, Farid El Oualid, Beatrix M. Ueberheide, Shaun K. Olsen, Tony T. Huang","doi":"10.1016/j.molcel.2025.10.028","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.028","url":null,"abstract":"In eukaryotes, each ribosomal subunit includes a ribosomal protein (RP) that is encoded as a fusion protein with ubiquitin (Ub). In yeast, each Ub-RP fusion requires processing by deubiquitylating enzymes (DUBs) to generate ribosome assembly-competent RPs and contribute to the cellular Ub pool. However, how Ub-RP fusions are processed by DUBs in human cells remains unclear. Here, we discovered that Ub-RPs are substrates of the Ub-fusion degradation (UFD) pathway in human cells via lysine 29 and 48 (K29/K48)-specific ubiquitylation and proteasomal degradation. We identified a pool of DUBs that catalytically process Ub-RPs, as well as DUBs that physically occlude Ub-RP interaction with UFD pathway Ub E3 ligases to prevent their degradation in a non-catalytic manner. Our results suggest that DUBs both process and stabilize Ub-RPs, whereas the UFD pathway regulates levels of Ub-RPs that cannot be fully processed by DUBs to fine-tune protein homeostasis.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"19 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145554812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-19DOI: 10.1016/j.molcel.2025.10.026
Brant Gracia, Xing-Han Zhang, Patricia Montes, Tin Chanh Pham, Min Huang, Junjie Chen, Georgios Ioannis Karras
Protein-folding chaperone heat shock protein 90 (HSP90) buffers genetic variation in diverse organisms, but the clinical significance of HSP90 buffering in human disease remains unclear. Here, we show that HSP90 buffers mutations in the BRCT domain of BRCA1. HSP90-buffered BRCA1 mutations result in protein variants that retain interactions with partner proteins and strongly rely on HSP90 for protein stability and function in cell survival. Moreover, HSP90-buffered BRCA1 variants confer poly (ADP-ribose) polymerase (PARP) inhibitor resistance in cancer cells. Low-level HSP90 inhibition overcomes this resistance, revealing a cryptic and mutant-specific HSP90-contingent synthetic lethality. Furthermore, by stabilizing metastable variants across the entirety of the BRCT domain, HSP90 reduces the clinical severity of BRCA1 mutations, allowing them to accumulate in populations. We estimate that HSP90 buffers 18% of known human BRCA1-BRCT missense mutations. Our work extends the clinical significance of HSP90 buffering to a prevalent class of variations in BRCA1, pioneering its importance in therapy resistance and cancer predisposition.
{"title":"HSP90 buffers deleterious genetic variations in BRCA1","authors":"Brant Gracia, Xing-Han Zhang, Patricia Montes, Tin Chanh Pham, Min Huang, Junjie Chen, Georgios Ioannis Karras","doi":"10.1016/j.molcel.2025.10.026","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.026","url":null,"abstract":"Protein-folding chaperone heat shock protein 90 (HSP90) buffers genetic variation in diverse organisms, but the clinical significance of HSP90 buffering in human disease remains unclear. Here, we show that HSP90 buffers mutations in the BRCT domain of BRCA1. HSP90-buffered <em>BRCA1</em> mutations result in protein variants that retain interactions with partner proteins and strongly rely on HSP90 for protein stability and function in cell survival. Moreover, HSP90-buffered BRCA1 variants confer poly (ADP-ribose) polymerase (PARP) inhibitor resistance in cancer cells. Low-level HSP90 inhibition overcomes this resistance, revealing a cryptic and mutant-specific HSP90-contingent synthetic lethality. Furthermore, by stabilizing metastable variants across the entirety of the BRCT domain, HSP90 reduces the clinical severity of <em>BRCA1</em> mutations, allowing them to accumulate in populations. We estimate that HSP90 buffers 18% of known human BRCA1-BRCT missense mutations. Our work extends the clinical significance of HSP90 buffering to a prevalent class of variations in <em>BRCA1</em>, pioneering its importance in therapy resistance and cancer predisposition.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"8 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145545665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-18DOI: 10.1016/j.molcel.2025.10.025
Rayees U.H. Mattoo, Dong-Hua Chen, David A. Bushnell, Sagi Tamir, Roger D. Kornberg
The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, a 1.8 MDa multi-subunit assembly comprising 19 subunits, is required for RNA polymerase II transcription in eukaryotes. The complex consists of four modules: transcription-associated protein 1 (Tra1), core, deubiquitination (DUB), and histone acetyltransferase (HAT). Although the structures of the Tra1, core, and DUB modules have been determined, the overall architecture of the HAT module remained elusive due to its inherent flexibility. To address this, we conducted cryo-electron microscopy (cryo-EM) analyses on SAGA purified from the thermophilic fungus Chaetomium thermophilum, yielding structures of Tra1 and core modules at 2.6 Å and three of the four HAT subunits at 3.7 Å. The structure of the HAT module was informative about the aspects of histone acetylation and the interface of HAT-core modules, contradicting earlier AlphaFold predictions. Our structure-guided genetic and biochemical analyses confirmed the roles of Ada1 and Spt7 in anchoring the HAT module within the SAGA complex.
{"title":"Structure of the transcriptional co-activator SAGA complex, including the histone acetyltransferase module","authors":"Rayees U.H. Mattoo, Dong-Hua Chen, David A. Bushnell, Sagi Tamir, Roger D. Kornberg","doi":"10.1016/j.molcel.2025.10.025","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.025","url":null,"abstract":"The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, a 1.8 MDa multi-subunit assembly comprising 19 subunits, is required for RNA polymerase II transcription in eukaryotes. The complex consists of four modules: transcription-associated protein 1 (Tra1), core, deubiquitination (DUB), and histone acetyltransferase (HAT). Although the structures of the Tra1, core, and DUB modules have been determined, the overall architecture of the HAT module remained elusive due to its inherent flexibility. To address this, we conducted cryo-electron microscopy (cryo-EM) analyses on SAGA purified from the thermophilic fungus <em>Chaetomium thermophilum</em>, yielding structures of Tra1 and core modules at 2.6 Å and three of the four HAT subunits at 3.7 Å. The structure of the HAT module was informative about the aspects of histone acetylation and the interface of HAT-core modules, contradicting earlier AlphaFold predictions. Our structure-guided genetic and biochemical analyses confirmed the roles of Ada1 and Spt7 in anchoring the HAT module within the SAGA complex.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"6 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145536473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Understanding the intricate relationship between three-dimensional chromatin structure and gene expression regulation is essential for cellular biology. However, current techniques are insufficient to capture regulatory elements functioning through three-dimensional chromatin structures. Here, we present high-throughput capture of actively transcribed region-interacting sequences (Hi-Coatis), a high-throughput method that seamlessly integrates the detection of active transcription signals with three-dimensional chromatin interaction studies. Hi-Coatis operates without antibodies or probes, enabling low-input cell experiments with high resolution and robustness, capturing more than 93% of expressed genes and over 60,000 regulatory loci in human cells. The repetitive/copy number variation (CNV) regions and the promoter regions of C2 genes, defined by the distribution patterns of Hi-Coatis signals, both exhibit strong regulatory element activity. Notably, in the Hemin-induced erythroid differentiation model of K562 cells, Hi-Coatis uncovers the potential for silent genes to transition to transcriptionally active states through the cooperative influence of specific transcription factors (e.g., CCCTC-binding factor [CTCF] and cohesin complex subunits Rad21 [RAD21]) and regulatory elements.
{"title":"High-throughput capture of actively transcribed region-interacting sequences reveals an intricate promoter-centered regulatory network","authors":"Xinxin Li, Jinsheng Xu, Xiaohao Yan, Jiayong Zhong, Chunhui Hou, Chuanle Xiao, Longjian Niu, Wei Chi","doi":"10.1016/j.molcel.2025.10.018","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.018","url":null,"abstract":"Understanding the intricate relationship between three-dimensional chromatin structure and gene expression regulation is essential for cellular biology. However, current techniques are insufficient to capture regulatory elements functioning through three-dimensional chromatin structures. Here, we present high-throughput capture of actively transcribed region-interacting sequences (Hi-Coatis), a high-throughput method that seamlessly integrates the detection of active transcription signals with three-dimensional chromatin interaction studies. Hi-Coatis operates without antibodies or probes, enabling low-input cell experiments with high resolution and robustness, capturing more than 93% of expressed genes and over 60,000 regulatory loci in human cells. The repetitive/copy number variation (CNV) regions and the promoter regions of C2 genes, defined by the distribution patterns of Hi-Coatis signals, both exhibit strong regulatory element activity. Notably, in the Hemin-induced erythroid differentiation model of K562 cells, Hi-Coatis uncovers the potential for silent genes to transition to transcriptionally active states through the cooperative influence of specific transcription factors (e.g., CCCTC-binding factor [CTCF] and cohesin complex subunits Rad21 [RAD21]) and regulatory elements.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"130 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145536092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1016/j.molcel.2025.10.019
Zhong Han, Shiqing Fu, Jens Vilstrup Johansen, David Lopez Martinez, Jiangman Lou, Thierry Boissière, Dandan He, Daniel Blears, Anouk M. Olthof, A. Barbara Dirac-Svejstrup, Jesper Q. Svejstrup
Senataxin (SETX) regulates RNA polymerase II (RNAPII) transcription and helps maintain genome stability, at least partly by suppressing R-loops. However, despite its importance in human disease, the precise function of SETX has remained unclear. Employing the degradation tag system for acute protein depletion, we demonstrate that SETX loss perturbs RNAPII elongation but does not markedly influence transcription termination at the end of genes. Through in vitro reconstitution of elongation, we show that SETX uses ATP-dependent RNA translocation to drive RNAPII forward across challenging DNA sequences, reminiscent of how bacterial ribosomes help mitigate RNAP pausing. In vivo, SETX depletion accordingly results in increased RNAPII pausing or backtracking, particularly during early elongation, with a corresponding, time-dependent local increase in R-loop formation. Together, these findings redefine our understanding of SETX’s role in transcription and provide a mechanistic framework for interpreting R-loops and the causes of neurological disorders associated with SETX mutation.
{"title":"A role for human senataxin in contending with pausing and backtracking during transcript elongation","authors":"Zhong Han, Shiqing Fu, Jens Vilstrup Johansen, David Lopez Martinez, Jiangman Lou, Thierry Boissière, Dandan He, Daniel Blears, Anouk M. Olthof, A. Barbara Dirac-Svejstrup, Jesper Q. Svejstrup","doi":"10.1016/j.molcel.2025.10.019","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.019","url":null,"abstract":"Senataxin (SETX) regulates RNA polymerase II (RNAPII) transcription and helps maintain genome stability, at least partly by suppressing R-loops. However, despite its importance in human disease, the precise function of SETX has remained unclear. Employing the degradation tag system for acute protein depletion, we demonstrate that SETX loss perturbs RNAPII elongation but does not markedly influence transcription termination at the end of genes. Through <em>in vitro</em> reconstitution of elongation, we show that SETX uses ATP-dependent RNA translocation to drive RNAPII forward across challenging DNA sequences, reminiscent of how bacterial ribosomes help mitigate RNAP pausing. <em>In vivo</em>, SETX depletion accordingly results in increased RNAPII pausing or backtracking, particularly during early elongation, with a corresponding, time-dependent local increase in R-loop formation. Together, these findings redefine our understanding of SETX’s role in transcription and provide a mechanistic framework for interpreting R-loops and the causes of neurological disorders associated with <em>SETX</em> mutation.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"102 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Since mitochondrial translation leads to the synthesis of the essential oxidative phosphorylation (OXPHOS) subunits, exhaustive and quantitative delineation of mitoribosome traversal is needed. Here, we developed a variety of high-resolution mitochondrial ribosome profiling derivatives and revealed the intricate regulation of mammalian mitochondrial translation. Harnessing a translation inhibitor, retapamulin, our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript, the elongation rate, and the initiation rate. We also surveyed the impacts of modifications at the anticodon stem loop in mitochondrial tRNAs (mt-tRNAs), including all possible modifications at the 34th position, in cells deleting the corresponding enzymes and derived from patients, as well as in mouse tissues. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed mitochondrial translation initiation factor (mtIF) 3-mediated translation initiation from internal open reading frames (ORFs) and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.
{"title":"Monitoring the complexity and dynamics of mitochondrial translation","authors":"Taisei Wakigawa, Mari Mito, Yushin Ando, Haruna Yamashiro, Kotaro Tomuro, Haruna Tani, Kazuhito Tomizawa, Takeshi Chujo, Asuteka Nagao, Takeo Suzuki, Osamu Nureki, Fan-Yan Wei, Yuichi Shichino, Yuzuru Itoh, Tsutomu Suzuki, Shintaro Iwasaki","doi":"10.1016/j.molcel.2025.10.022","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.022","url":null,"abstract":"Since mitochondrial translation leads to the synthesis of the essential oxidative phosphorylation (OXPHOS) subunits, exhaustive and quantitative delineation of mitoribosome traversal is needed. Here, we developed a variety of high-resolution mitochondrial ribosome profiling derivatives and revealed the intricate regulation of mammalian mitochondrial translation. Harnessing a translation inhibitor, retapamulin, our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript, the elongation rate, and the initiation rate. We also surveyed the impacts of modifications at the anticodon stem loop in mitochondrial tRNAs (mt-tRNAs), including all possible modifications at the 34th position, in cells deleting the corresponding enzymes and derived from patients, as well as in mouse tissues. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed mitochondrial translation initiation factor (mtIF) 3-mediated translation initiation from internal open reading frames (ORFs) and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"86 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-07DOI: 10.1016/j.molcel.2025.10.015
Samantha B. Regan, Darpan Medhi, Yuanlin Xu, Travis B. White, Yi-Zhen Jiang, Jung Eun Kim, Shih-Chun Wang, Qichen Deng, Su Jia, Dulguun Baasan, Jon P. Connelly, Ti-Cheng Chang, Shondra M. Pruett-Miller, Maria Jasin
Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.
{"title":"Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks","authors":"Samantha B. Regan, Darpan Medhi, Yuanlin Xu, Travis B. White, Yi-Zhen Jiang, Jung Eun Kim, Shih-Chun Wang, Qichen Deng, Su Jia, Dulguun Baasan, Jon P. Connelly, Ti-Cheng Chang, Shondra M. Pruett-Miller, Maria Jasin","doi":"10.1016/j.molcel.2025.10.015","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.015","url":null,"abstract":"Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with <em>de novo</em> telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"10 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145454666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-07DOI: 10.1016/j.molcel.2025.10.013
Jimmy Ly, Matteo Di Bernardo, Yi Fei Tao, Ekaterina Khalizeva, Christopher J. Giuliano, Sebastian Lourido, Mark D. Fleming, Iain M. Cheeseman
Rare genetic diseases collectively affect millions of individuals. A common target of many rare diseases is the mitochondria, intracellular organelles that originated through endosymbiosis. Eukaryotic cells require related proteins to function both within the mitochondria and in the host cell. By analyzing N-terminal protein isoforms generated through alternative start codon selection, we identify hundreds of differentially localized isoform pairs, including dual-localized isoforms that are essential for both mitochondrial and host cell function. Subsets of dual mitochondria-localized isoforms emerged during early eukaryotic evolution, coinciding with mitochondrial endosymbiosis. Importantly, we identify dozens of rare disease alleles that affect these alternative protein variants with unique molecular and clinical consequences. Alternative start codon selection can bypass pathogenic nonsense and frameshift mutations, thereby selectively eliminating specific isoforms, which we term isoform-selective alleles (ISAs). Together, our findings illuminate the evolutionary and pathological relevance of alternative translation, offering insights into the molecular basis of rare human diseases.
{"title":"Alternative start codon selection shapes mitochondrial function and rare human diseases","authors":"Jimmy Ly, Matteo Di Bernardo, Yi Fei Tao, Ekaterina Khalizeva, Christopher J. Giuliano, Sebastian Lourido, Mark D. Fleming, Iain M. Cheeseman","doi":"10.1016/j.molcel.2025.10.013","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.013","url":null,"abstract":"Rare genetic diseases collectively affect millions of individuals. A common target of many rare diseases is the mitochondria, intracellular organelles that originated through endosymbiosis. Eukaryotic cells require related proteins to function both within the mitochondria and in the host cell. By analyzing N-terminal protein isoforms generated through alternative start codon selection, we identify hundreds of differentially localized isoform pairs, including dual-localized isoforms that are essential for both mitochondrial and host cell function. Subsets of dual mitochondria-localized isoforms emerged during early eukaryotic evolution, coinciding with mitochondrial endosymbiosis. Importantly, we identify dozens of rare disease alleles that affect these alternative protein variants with unique molecular and clinical consequences. Alternative start codon selection can bypass pathogenic nonsense and frameshift mutations, thereby selectively eliminating specific isoforms, which we term isoform-selective alleles (ISAs). Together, our findings illuminate the evolutionary and pathological relevance of alternative translation, offering insights into the molecular basis of rare human diseases.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"92 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145454663","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-06Epub Date: 2025-10-20DOI: 10.1016/j.molcel.2025.09.030
Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, Sae-Hun Park, Nikola Wagener, Anne Devin, Christof Osman, Walter Neupert, Dejana Mokranjac
Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.
{"title":"Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.","authors":"Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, Sae-Hun Park, Nikola Wagener, Anne Devin, Christof Osman, Walter Neupert, Dejana Mokranjac","doi":"10.1016/j.molcel.2025.09.030","DOIUrl":"10.1016/j.molcel.2025.09.030","url":null,"abstract":"<p><p>Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.</p>","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":" ","pages":"4082-4092.e7"},"PeriodicalIF":16.6,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145346180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-06DOI: 10.1016/j.molcel.2025.10.016
Anthony R.P. Verkerke, Shingo Kajimura
In a recent publication in Nature, Liu et al.1 report a UCP1-independent thermogenic mechanism in which peroxisomes generate heat in brown adipose tissue through active synthesis and oxidation of monomethyl branched-chain fatty acids (mmBCFAs) derived from branched-chain amino acids.
{"title":"A new UCP1-independent thermogenic mechanism in peroxisomes","authors":"Anthony R.P. Verkerke, Shingo Kajimura","doi":"10.1016/j.molcel.2025.10.016","DOIUrl":"https://doi.org/10.1016/j.molcel.2025.10.016","url":null,"abstract":"In a recent publication in <em>Nature</em>, Liu et al.<span><span><sup>1</sup></span></span> report a UCP1-independent thermogenic mechanism in which peroxisomes generate heat in brown adipose tissue through active synthesis and oxidation of monomethyl branched-chain fatty acids (mmBCFAs) derived from branched-chain amino acids.","PeriodicalId":18950,"journal":{"name":"Molecular Cell","volume":"97 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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