Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611646
Alicia Santos-Aledo, Adrian Plaza-Pegueroles, Marta Sanz-Murillo, Federico M. Ruiz, Jun Xu, David Gil-Carton, Dong Wang, Carlos Fernandez-Tornero
RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites slow nucleotide, which occurs by addition by base sandwiching between the RNA 3-prime end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe route to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.
RNA 聚合酶 I（Pol I）转录核糖体 DNA（rDNA）以产生 rRNA 前体，它占生长细胞总转录活性的 60%。Pol I 监控着 rDNA 的完整性并影响着细胞的存活，但人们对这种酶如何处理消融 DNA 病变却知之甚少。在此，我们报告了 Pol I 在消减位点停滞的不同阶段的电子低温显微镜（cryo-EM）结构，并辅以体外转录研究。我们的研究结果表明，模板化消融位点会减慢核苷酸的速度，这是由 RNA 3-prime末端和Pol I桥螺旋之间的碱基夹层加成的。然而，模板化消融位点的存在会促使 Pol I 裂隙打开，使酶从 DNA 中解离，或使 A12-Ct 进入活性位点，从而刺激 RNA 的裂解。与病变相对的核苷酸添加会诱发早期易位中间体，这种中间体与之前描述的 RNA 聚合酶暂停状态不同，因为杂交体中的 DNA 碱基会倾斜，与新添加的 RNA 碱基形成氢键。在这种状态下，核苷酸的添加是极不受欢迎的，而固有的 Pol I RNA 裂解活性则充当了将病变旁路最小化的故障安全路径。我们的研究结果揭示了核苷酸添加到 Ap 位点对面后导致 Pol I 持续停滞的两步机制，这种停滞不同于 CPD 病变的停滞，也不同于 Pol II 在 Ap 位点的阻滞。
{"title":"Cryo-EM uncovers a sequential mechanism for RNA polymerase I pausing and stalling at abasic DNA lesions","authors":"Alicia Santos-Aledo, Adrian Plaza-Pegueroles, Marta Sanz-Murillo, Federico M. Ruiz, Jun Xu, David Gil-Carton, Dong Wang, Carlos Fernandez-Tornero","doi":"10.1101/2024.09.09.611646","DOIUrl":"https://doi.org/10.1101/2024.09.09.611646","url":null,"abstract":"RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites slow nucleotide, which occurs by addition by base sandwiching between the RNA 3-prime end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe route to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"98 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612099
Luis F Ceriotti, Jessica M Warren, M. Virginia Sanchez-Puerta, Daniel B Sloan
The function of tRNAs depends on enzymes that cleave primary transcript ends, add a 3' CCA tail, introduce post-transcriptional base modifications, and charge (aminoacylate) mature tRNAs with the correct amino acid. Maintaining an available pool of the resulting aminoacylated tRNAs is essential for protein synthesis. High-throughput sequencing techniques have recently been developed to provide a comprehensive view of aminoacylation state in a tRNA-specific fashion. However, these methods have never been applied to plants. Here, we treated Arabidopsis thaliana RNA samples with periodate and then performed tRNA-seq to distinguish between aminoacylated and uncharged tRNAs. This approach successfully captured every tRNA isodecoder family and detected expression of additional tRNA-like transcripts. We found that estimated aminoacylation rates and CCA tail integrity were significantly higher on average for organellar (mitochondrial and plastid) tRNAs than for nuclear/cytosolic tRNAs. Reanalysis of previously published human cell line data showed a similar pattern. Base modifications result in nucleotide misincorporations and truncations during reverse transcription, which we quantified and used to test for relationships with aminoacylation levels. We also determined that the Arabidopsis tRNA-like sequences (t-elements) that are cleaved from the ends of some mitochondrial mRNAs have post-transcriptionally modified bases and CCA-tail addition. However, these t-elements are not aminoacylated, indicating that they are only recognized by a subset of tRNA-interacting enzymes and do not play a role in translation. Overall, this work provides a characterization of the baseline landscape of plant tRNA aminoacylation rates and demonstrates an approach for investigating environmental and genetic perturbations to plant translation machinery.
{"title":"The landscape of Arabidopsis tRNA aminoacylation","authors":"Luis F Ceriotti, Jessica M Warren, M. Virginia Sanchez-Puerta, Daniel B Sloan","doi":"10.1101/2024.09.09.612099","DOIUrl":"https://doi.org/10.1101/2024.09.09.612099","url":null,"abstract":"The function of tRNAs depends on enzymes that cleave primary transcript ends, add a 3' CCA tail, introduce post-transcriptional base modifications, and charge (aminoacylate) mature tRNAs with the correct amino acid. Maintaining an available pool of the resulting aminoacylated tRNAs is essential for protein synthesis. High-throughput sequencing techniques have recently been developed to provide a comprehensive view of aminoacylation state in a tRNA-specific fashion. However, these methods have never been applied to plants. Here, we treated Arabidopsis thaliana RNA samples with periodate and then performed tRNA-seq to distinguish between aminoacylated and uncharged tRNAs. This approach successfully captured every tRNA isodecoder family and detected expression of additional tRNA-like transcripts. We found that estimated aminoacylation rates and CCA tail integrity were significantly higher on average for organellar (mitochondrial and plastid) tRNAs than for nuclear/cytosolic tRNAs. Reanalysis of previously published human cell line data showed a similar pattern. Base modifications result in nucleotide misincorporations and truncations during reverse transcription, which we quantified and used to test for relationships with aminoacylation levels. We also determined that the Arabidopsis tRNA-like sequences (t-elements) that are cleaved from the ends of some mitochondrial mRNAs have post-transcriptionally modified bases and CCA-tail addition. However, these t-elements are not aminoacylated, indicating that they are only recognized by a subset of tRNA-interacting enzymes and do not play a role in translation. Overall, this work provides a characterization of the baseline landscape of plant tRNA aminoacylation rates and demonstrates an approach for investigating environmental and genetic perturbations to plant translation machinery.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"178 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611872
Zoya A. Gauhar, Cameron Myhrvold, Elizabeth R. Gavis
Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila, the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nos) and sarah (sra), whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observe defective primordial germ cell migration, and an increase in the calcium indicator GCaMP at the posterior, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.
{"title":"Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs","authors":"Zoya A. Gauhar, Cameron Myhrvold, Elizabeth R. Gavis","doi":"10.1101/2024.09.09.611872","DOIUrl":"https://doi.org/10.1101/2024.09.09.611872","url":null,"abstract":"Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila, the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nos) and sarah (sra), whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observe defective primordial germ cell migration, and an increase in the calcium indicator GCaMP at the posterior, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612072
Pranay Ramteke, Bahiyah Watson, Mallory Toci, Victoria Tran, Shira Johnston, Maria Tsingas, Ruteja Barve, Ramkrishna Mitra, Richard Loeser, John Collins, Makarand Risbud
Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (AcanCreERT2; Sirt6fl/fl). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, yH2AX, IL-6, and TGF-b; abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.
{"title":"SIRT6 loss causes intervertebral disc degeneration in mice by promoting senescence and SASP status","authors":"Pranay Ramteke, Bahiyah Watson, Mallory Toci, Victoria Tran, Shira Johnston, Maria Tsingas, Ruteja Barve, Ramkrishna Mitra, Richard Loeser, John Collins, Makarand Risbud","doi":"10.1101/2024.09.09.612072","DOIUrl":"https://doi.org/10.1101/2024.09.09.612072","url":null,"abstract":"Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (AcanCreERT2; Sirt6fl/fl). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, yH2AX, IL-6, and TGF-b; abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612016
Eric W Deutsch, Leron W Kok, Jonathan M Mudge, Jorge Ruiz-Orera, Ivo Fierro-Monti, Zhi Sun, Jennifer G Abelin, M Mar Alba, Julie L Aspden, Ariel A Bazzini, Elspeth Bruford, Marie A Brunet, Lorenzo Calviello, Steven A Carr, Anne-Ruxandra Carvunis, Sonia Chothani, Jim Clauwaert, Kellie Dean, Pouya Faridi, Adam Frankish, Norbert Hubner, Nicholas Ingolia, Michele Magrane, Maria Jesus Martin, Thomas F Martinez, Gerben Menschaert, Uwe Ohler, Sandra Orchard, Owen Rackham, Xavier Roucou, Sarah A Slavoff, Eivind Valen, Aaron C Wacholder, Jonathan S. Weissman, Wei Wu, Zhi Xie, Jyoti Choudhary, Michal Bassani-Sternberg, Juan Antonio Vizcaino, Nicola Ternette, Robert L. Moritz, John Prensner, Sebastiaan van Heesch
A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.
{"title":"High-quality peptide evidence for annotating non-canonical open reading frames as human proteins","authors":"Eric W Deutsch, Leron W Kok, Jonathan M Mudge, Jorge Ruiz-Orera, Ivo Fierro-Monti, Zhi Sun, Jennifer G Abelin, M Mar Alba, Julie L Aspden, Ariel A Bazzini, Elspeth Bruford, Marie A Brunet, Lorenzo Calviello, Steven A Carr, Anne-Ruxandra Carvunis, Sonia Chothani, Jim Clauwaert, Kellie Dean, Pouya Faridi, Adam Frankish, Norbert Hubner, Nicholas Ingolia, Michele Magrane, Maria Jesus Martin, Thomas F Martinez, Gerben Menschaert, Uwe Ohler, Sandra Orchard, Owen Rackham, Xavier Roucou, Sarah A Slavoff, Eivind Valen, Aaron C Wacholder, Jonathan S. Weissman, Wei Wu, Zhi Xie, Jyoti Choudhary, Michal Bassani-Sternberg, Juan Antonio Vizcaino, Nicola Ternette, Robert L. Moritz, John Prensner, Sebastiaan van Heesch","doi":"10.1101/2024.09.09.612016","DOIUrl":"https://doi.org/10.1101/2024.09.09.612016","url":null,"abstract":"A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"39 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.08.611865
Michael T Forrester, Jacob R. Egol, Sinan Ozbay, Rohit Singh, Purushothama Rao Tata
Protein S-palmitoylation is a reversible lipophilic posttranslational modification regulating a diverse number of signaling pathways. Within transmembrane proteins (TMPs), S-palmitoylation is implicated in conditions from inflammatory disorders to respiratory viral infections. Many small-scale experiments have observed S-palmitoylation at juxtamembrane Cys residues. However, most large-scale S-palmitoyl discovery efforts rely on trypsin-based proteomics within which hydrophobic juxtamembrane regions are likely underrepresented. Machine learning, by virtue of its freedom from experimental constraints, is particularly well suited to address this discovery gap surrounding TMP S-palmitoylation. Utilizing a UniProt-derived feature set, a gradient boosted machine learning tool (TopoPalmTree) was constructed and applied to a holdout dataset of viral S-palmitoylated proteins. Upon application to the mouse TMP proteome, 1591 putative S-palmitoyl sites (i.e. not listed in SwissPalm or UniProt) were identified. Two lung-expressed S-palmitoyl candidates (synaptobrevin Vamp5 and water channel Aquaporin-5) were experimentally assessed. Finally, TopoPalmTree was used for rational design of an S-palmitoyl site on KDEL-Receptor 2. This readily interpretable model aligns the innumerable small-scale experiments observing juxtamembrane S-palmitoylation into a proteomic tool for TMP S-palmitoyl discovery and design, thus facilitating future investigations of this important modification.
{"title":"Topology-Driven Discovery of Transmembrane Protein S-Palmitoylation","authors":"Michael T Forrester, Jacob R. Egol, Sinan Ozbay, Rohit Singh, Purushothama Rao Tata","doi":"10.1101/2024.09.08.611865","DOIUrl":"https://doi.org/10.1101/2024.09.08.611865","url":null,"abstract":"Protein <em>S</em>-palmitoylation is a reversible lipophilic posttranslational modification regulating a diverse number of signaling pathways. Within transmembrane proteins (TMPs), <em>S</em>-palmitoylation is implicated in conditions from inflammatory disorders to respiratory viral infections. Many small-scale experiments have observed <em>S</em>-palmitoylation at juxtamembrane Cys residues. However, most large-scale <em>S</em>-palmitoyl discovery efforts rely on trypsin-based proteomics within which hydrophobic juxtamembrane regions are likely underrepresented. Machine learning, by virtue of its freedom from experimental constraints, is particularly well suited to address this discovery gap surrounding TMP <em>S</em>-palmitoylation. Utilizing a UniProt-derived feature set, a gradient boosted machine learning tool (TopoPalmTree) was constructed and applied to a holdout dataset of viral <em>S</em>-palmitoylated proteins. Upon application to the mouse TMP proteome, 1591 putative <em>S</em>-palmitoyl sites (i.e. not listed in SwissPalm or UniProt) were identified. Two lung-expressed <em>S</em>-palmitoyl candidates (synaptobrevin Vamp5 and water channel Aquaporin-5) were experimentally assessed. Finally, TopoPalmTree was used for rational design of an <em>S</em>-palmitoyl site on KDEL-Receptor 2. This readily interpretable model aligns the innumerable small-scale experiments observing juxtamembrane <em>S</em>-palmitoylation into a proteomic tool for TMP <em>S</em>-palmitoyl discovery and design, thus facilitating future investigations of this important modification.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212815","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.08.611871
Pablo David Jimenez Castro, Christian Leutenegger, Christian Savard, Cecilia Lozoya, Holly Brown, Jennifer Willcox, Haresh Rochani, Heather Martinez
Ancylostoma caninum is the most significant intestinal nematode parasite of dogs. We acquired fecal surveillance data using molecular diagnostics in a large population of dogs in the United States (US). A diagnostic test using real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting the SNP F167Y was used in 885,424 canine fecal samples collected between March 2022 and December 2023. Overall, Ancylostoma spp. had a prevalence of 1.76% (15,537/885,424), with the highest observed in the South 3.73% (10,747/287,576), and the lowest in the West 0.45% (632/140,282). Within the subset of Ancylostoma spp.-detected dogs used for further analysis, the F167Y SNP had an overall prevalence of 14.2% with the highest in the West and the lowest in the Midwest (10.76%). The greyhound breed exhibited a higher prevalence of Ancylostoma spp. infections (17.03%) and a higher prevalence of the F167Y polymorphism (33.6%) compared to non-greyhound breeds (13.7% and 2.08%), respectively, but were not associated with the highest breed risk for the F167Y polymorphism. Sex did not influence hookworm infection nor F167Y polymorphism prevalence. Intact dogs had a prevalence of hookworm infection and F167Y polymorphism of 2.51% and 14.6%, respectively. Puppies showed increased prevalence of hookworms (3.70%) and the F167Y SNP (17.1%). Greyhounds, bluetick coonhounds, and boerboels had the highest relative risks (RR) for hookworm infection, while Cavalier King Charles spaniels, Havanese, and shiba inus had the lowest. The top and bottom three with the highest and lowest RR for the F167Y SNP were the old English sheepdog, American foxhound, and toy poodle Toy, and shih tzu, Maltese, and Australian cattle dogs, respectively. This study highlights the value of an accessible diagnostic qPCR test with fast turnaround times in unraveling the molecular epidemiology of hookworms and benzimidazole resistance, as well as explore potentially important risk factors associated with infection in medicalized dogs.
{"title":"Investigation of risk factors associated with Ancylostoma spp. infection and the benzimidazole F167Y resistance marker polymorphism in dogs from the United States","authors":"Pablo David Jimenez Castro, Christian Leutenegger, Christian Savard, Cecilia Lozoya, Holly Brown, Jennifer Willcox, Haresh Rochani, Heather Martinez","doi":"10.1101/2024.09.08.611871","DOIUrl":"https://doi.org/10.1101/2024.09.08.611871","url":null,"abstract":"Ancylostoma caninum is the most significant intestinal nematode parasite of dogs. We acquired fecal surveillance data using molecular diagnostics in a large population of dogs in the United States (US). A diagnostic test using real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting the SNP F167Y was used in 885,424 canine fecal samples collected between March 2022 and December 2023. Overall, Ancylostoma spp. had a prevalence of 1.76% (15,537/885,424), with the highest observed in the South 3.73% (10,747/287,576), and the lowest in the West 0.45% (632/140,282). Within the subset of Ancylostoma spp.-detected dogs used for further analysis, the F167Y SNP had an overall prevalence of 14.2% with the highest in the West and the lowest in the Midwest (10.76%). The greyhound breed exhibited a higher prevalence of Ancylostoma spp. infections (17.03%) and a higher prevalence of the F167Y polymorphism (33.6%) compared to non-greyhound breeds (13.7% and 2.08%), respectively, but were not associated with the highest breed risk for the F167Y polymorphism. Sex did not influence hookworm infection nor F167Y polymorphism prevalence. Intact dogs had a prevalence of hookworm infection and F167Y polymorphism of 2.51% and 14.6%, respectively. Puppies showed increased prevalence of hookworms (3.70%) and the F167Y SNP (17.1%). Greyhounds, bluetick coonhounds, and boerboels had the highest relative risks (RR) for hookworm infection, while Cavalier King Charles spaniels, Havanese, and shiba inus had the lowest. The top and bottom three with the highest and lowest RR for the F167Y SNP were the old English sheepdog, American foxhound, and toy poodle Toy, and shih tzu, Maltese, and Australian cattle dogs, respectively. This study highlights the value of an accessible diagnostic qPCR test with fast turnaround times in unraveling the molecular epidemiology of hookworms and benzimidazole resistance, as well as explore potentially important risk factors associated with infection in medicalized dogs.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.07.611797
Soren Warming, Zhenyi Liu, Ming-Chi Tsai, Soumitra Ghosh, Jessica Lawrence, Sarah Chu, Baris Bingol
Targeting of proteins for degradation in a reversible manner is a powerful approach to decipher gene function and mimic drug effects, with great potential for drug target discovery and validation. A generalized approach is to tag a protein of interest and then use this tag to recruit an endogenously or exogenously expressed E3 ligase for its polyubiquitination and subsequent degradation via 26S proteasome. However, the often bulky size of the tag and the great variability of substrate-dependent degradation efficiency of mammalian E3 ligases pose great challenges in practice. Here we show that small tags (10-15 amino acids) can be used to efficiently tag endogenous proteins for degradation when coupled with an exogenously expressed artificial bacterial E3 ligase (ABEL) consisting of a tag-interacting moiety and the catalytic domain of the bacterial E3 ligase IpaH9.8. We name this versatile and efficient platform degradation by small tag ABEL (DESTABEL). Furthermore, we show that an ABEL containing a nanobody against human α-synuclein mediates efficient degradation in primary neurons as well as in the adult mouse brain. Taken together, our data show that tag-dependent and independent ABELs are powerful yet flexible tools for studies of protein function and drug target validation.
以可逆方式靶向降解蛋白质是破译基因功能和模拟药物效应的有力方法，在药物靶点发现和验证方面具有巨大潜力。一种通用的方法是标记感兴趣的蛋白质，然后利用该标记招募内源性或外源性表达的 E3 连接酶，使其多泛素化，随后通过 26S 蛋白酶体降解。然而，标签通常体积庞大，哺乳动物 E3 连接酶依赖底物的降解效率差异很大，这给实际操作带来了巨大挑战。在这里，我们展示了当小标签（10-15 个氨基酸）与外源表达的人工细菌 E3 连接酶（ABEL）结合时，可以有效地标记内源性蛋白质进行降解。ABEL 由一个标签连接分子和细菌 E3 连接酶 IpaH9.8 的催化结构域组成。我们将这一多功能高效平台命名为小标签 ABEL 降解（DESTABEL）。此外，我们还表明，含有针对人类α-突触核蛋白的纳米抗体的ABEL能在原代神经元和成年小鼠大脑中介导高效降解。总之，我们的数据表明，标签依赖型和独立 ABEL 是研究蛋白质功能和验证药物靶点的强大而灵活的工具。
{"title":"Protein degradation by small tag artificial bacterial E3 ligase","authors":"Soren Warming, Zhenyi Liu, Ming-Chi Tsai, Soumitra Ghosh, Jessica Lawrence, Sarah Chu, Baris Bingol","doi":"10.1101/2024.09.07.611797","DOIUrl":"https://doi.org/10.1101/2024.09.07.611797","url":null,"abstract":"Targeting of proteins for degradation in a reversible manner is a powerful approach to decipher gene function and mimic drug effects, with great potential for drug target discovery and validation. A generalized approach is to tag a protein of interest and then use this tag to recruit an endogenously or exogenously expressed E3 ligase for its polyubiquitination and subsequent degradation via 26S proteasome. However, the often bulky size of the tag and the great variability of substrate-dependent degradation efficiency of mammalian E3 ligases pose great challenges in practice. Here we show that small tags (10-15 amino acids) can be used to efficiently tag endogenous proteins for degradation when coupled with an exogenously expressed artificial bacterial E3 ligase (ABEL) consisting of a tag-interacting moiety and the catalytic domain of the bacterial E3 ligase IpaH9.8. We name this versatile and efficient platform degradation by small tag ABEL (DESTABEL). Furthermore, we show that an ABEL containing a nanobody against human α-synuclein mediates efficient degradation in primary neurons as well as in the adult mouse brain. Taken together, our data show that tag-dependent and independent ABELs are powerful yet flexible tools for studies of protein function and drug target validation.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"273 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.07.611803
Shimul Ghosh, Aaron W Bivins
Digital PCR (dPCR) shows great promise for precise, sensitive, and inhibition-resilient measurement of nucleic acids in surface water, providing an advantage for applications such as microbial source tracking (MST). Herein, we describe our empirical optimization of two triplex format dPCR reactions (Triplex 1 - Cow M2, Rum2Bac, Cow M3; Triplex 2 - HF183/BacR287, Pig2Bac, Entero1a) on the QIAcuity One system for MST. Each triplex produced gene copy measurements similar to single-plex format assays for a standard reference material (SRM 2917) at a fixed concentration and along a concentration gradient. For achieved water samples previously tested by single-plex assays, each triplex also produced MST marker measurements comparable to the single-plex results. The triplexes described here can be directly adopted for MST on the QIAcuity, or the optimization protocol we demonstrate can be used to develop additional multiplex assays on the QIAcuity system.
{"title":"Optimizing Digital PCR Assays for Multiplexed Microbial Source Tracking in Surface Waters","authors":"Shimul Ghosh, Aaron W Bivins","doi":"10.1101/2024.09.07.611803","DOIUrl":"https://doi.org/10.1101/2024.09.07.611803","url":null,"abstract":"Digital PCR (dPCR) shows great promise for precise, sensitive, and inhibition-resilient measurement of nucleic acids in surface water, providing an advantage for applications such as microbial source tracking (MST). Herein, we describe our empirical optimization of two triplex format dPCR reactions (Triplex 1 - Cow M2, Rum2Bac, Cow M3; Triplex 2 - HF183/BacR287, Pig2Bac, Entero1a) on the QIAcuity One system for MST. Each triplex produced gene copy measurements similar to single-plex format assays for a standard reference material (SRM 2917) at a fixed concentration and along a concentration gradient. For achieved water samples previously tested by single-plex assays, each triplex also produced MST marker measurements comparable to the single-plex results. The triplexes described here can be directly adopted for MST on the QIAcuity, or the optimization protocol we demonstrate can be used to develop additional multiplex assays on the QIAcuity system.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.06.611206
Roni Klein, Janset Onyuru, Estela M. Viera, Christopher D. Putnam, Hal M. Hoffman, Michelle L. Hastings
Inflammation has an essential role in healing. However, over-active inflammation disrupts normal cellular functions and can be life-threatening when not resolved. The NLRP3 inflammasome, a component of the innate immune system, is an intracellular multiprotein complex that senses stress-associated signals, and, for this reason is a promising therapeutic target for treating unresolved, pathogenic inflammation. Alternative splicing of NLRP3 RNA has been suggested as a regulatory mechanism for inflammasome activation, as some spliced isoforms encode NLRP3 proteins with compromised function. Here, we take advantage of this natural regulatory mechanism and devise a way to control pathogenic inflammation using splice-switching antisense oligonucleotides (ASOs). To identify and induce NLRP3 spliced isoforms lacking inflammatory activity, we tested a series of ASOs, each targeting a different exon, to determine the most effective strategy for down-regulating NLRP3. We identify several ASOs that modulate NLRP3 splicing, reduce NLRP3 protein, and decrease inflammasome signaling in vitro. The most effective ASO suppresses systemic inflammation in vivo in mouse models of acute inflammation and cryopyrin-associated periodic syndrome (CAPS). Our results demonstrate a systematic approach to protein engineering using splice-switching ASOs to generate isoforms with altered activity, and identify an ASO that can treat pathological inflammation in mice by reducing functional NLRP3.
{"title":"Modulating NLRP3 splicing with antisense oligonucleotides to control pathological inflammation","authors":"Roni Klein, Janset Onyuru, Estela M. Viera, Christopher D. Putnam, Hal M. Hoffman, Michelle L. Hastings","doi":"10.1101/2024.09.06.611206","DOIUrl":"https://doi.org/10.1101/2024.09.06.611206","url":null,"abstract":"Inflammation has an essential role in healing. However, over-active inflammation disrupts normal cellular functions and can be life-threatening when not resolved. The NLRP3 inflammasome, a component of the innate immune system, is an intracellular multiprotein complex that senses stress-associated signals, and, for this reason is a promising therapeutic target for treating unresolved, pathogenic inflammation. Alternative splicing of NLRP3 RNA has been suggested as a regulatory mechanism for inflammasome activation, as some spliced isoforms encode NLRP3 proteins with compromised function. Here, we take advantage of this natural regulatory mechanism and devise a way to control pathogenic inflammation using splice-switching antisense oligonucleotides (ASOs). To identify and induce NLRP3 spliced isoforms lacking inflammatory activity, we tested a series of ASOs, each targeting a different exon, to determine the most effective strategy for down-regulating NLRP3. We identify several ASOs that modulate NLRP3 splicing, reduce NLRP3 protein, and decrease inflammasome signaling in vitro. The most effective ASO suppresses systemic inflammation in vivo in mouse models of acute inflammation and cryopyrin-associated periodic syndrome (CAPS). Our results demonstrate a systematic approach to protein engineering using splice-switching ASOs to generate isoforms with altered activity, and identify an ASO that can treat pathological inflammation in mice by reducing functional NLRP3.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"52 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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