Pub Date : 2024-09-17DOI: 10.1101/2024.09.17.613553
Jackson A Hoffman, Kevin W Trotter, Trevor K. Archer
Nucleosomes at actively transcribed promoters have specific histone post-transcriptional modifications and histone variants. These features are thought to contribute to the formation and maintenance of a permissive chromatin environment. Recent reports have drawn conflicting conclusions about whether these histone modifications depend on transcription. We used triptolide to inhibit transcription initiation and degrade RNA Polymerase II and interrogated the effect on histone modifications. Transcription initiation was dispensable for de novo and steady-state histone acetylation at transcription start sites (TSSs) and enhancers. However, at steady state, blocking transcription initiation increased the levels of histone acetylation and H2AZ incorporation at active TSSs. These results demonstrate that deposition of specific histone modifications at TSSs is not dependent on transcription and that transcription limits the maintenance of these marks.
{"title":"RNA Polymerase II coordinates histone deacetylation at active promoters","authors":"Jackson A Hoffman, Kevin W Trotter, Trevor K. Archer","doi":"10.1101/2024.09.17.613553","DOIUrl":"https://doi.org/10.1101/2024.09.17.613553","url":null,"abstract":"Nucleosomes at actively transcribed promoters have specific histone post-transcriptional modifications and histone variants. These features are thought to contribute to the formation and maintenance of a permissive chromatin environment. Recent reports have drawn conflicting conclusions about whether these histone modifications depend on transcription. We used triptolide to inhibit transcription initiation and degrade RNA Polymerase II and interrogated the effect on histone modifications. Transcription initiation was dispensable for de novo and steady-state histone acetylation at transcription start sites (TSSs) and enhancers. However, at steady state, blocking transcription initiation increased the levels of histone acetylation and H2AZ incorporation at active TSSs. These results demonstrate that deposition of specific histone modifications at TSSs is not dependent on transcription and that transcription limits the maintenance of these marks.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268929","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}
ALS, Amyotrophic lateral sclerosis, a devastating neurodegenerative disease (ND) with no cure, is often caused by abnormal cytosolic aggregation of RNA-binding proteins, the most well-known of which are TDP-43 and FUS. The proteasome is considered one of the major systems that degrades misfolded, including ND-associated, proteins, thereby acting to reduce aggregation, while inhibition of the proteasome increases aggregation. Unexpectedly, we found that proteasome inhibitor treatment significantly reduced ALS-associated mutant FUS aggregation in cells and in primary neurons. This is in sharp contrast to most other ND-associated aggregating proteins, including Huntingtin and TDP-43, for which proteasome inhibitors enhanced aggregation. We further found that this inhibitory effect is dependent on the transcription factor HSF1, suggesting that the underlying mechanism of this effect is transcriptionally-mediated. Since heat shock treatment did not show any effect on FUS aggregation, we hypothesized that proteasome inhibitors elicit a transcriptional program distinct of that of heat shock, which is protective of FUS aggregation. We identified BAG3, a co-chaperone that cooperates with HSP70 in reducing FUS aggregation, as a significant mediator of this effect. We therefore propose BBB-permeable proteasome inhibitors as a potential therapy specific to ALS-FUS.
{"title":"Targeting FUS-ALS aggregation with Proteasome Inhibitors","authors":"Amal Younis, Kanar Yassen, Kinneret Rozales, Tahani Kadah, Naseeb Saida, Anatoly Meller, Joyeeta Dutta Hazra, Ronit Heinrich, Flonia Levy-Adam, Shai Berlin, Reut Shalgi","doi":"10.1101/2024.09.17.613412","DOIUrl":"https://doi.org/10.1101/2024.09.17.613412","url":null,"abstract":"ALS, Amyotrophic lateral sclerosis, a devastating neurodegenerative disease (ND) with no cure, is often caused by abnormal cytosolic aggregation of RNA-binding proteins, the most well-known of which are TDP-43 and FUS. The proteasome is considered one of the major systems that degrades misfolded, including ND-associated, proteins, thereby acting to reduce aggregation, while inhibition of the proteasome increases aggregation. Unexpectedly, we found that proteasome inhibitor treatment significantly reduced ALS-associated mutant FUS aggregation in cells and in primary neurons. This is in sharp contrast to most other ND-associated aggregating proteins, including Huntingtin and TDP-43, for which proteasome inhibitors enhanced aggregation. We further found that this inhibitory effect is dependent on the transcription factor HSF1, suggesting that the underlying mechanism of this effect is transcriptionally-mediated. Since heat shock treatment did not show any effect on FUS aggregation, we hypothesized that proteasome inhibitors elicit a transcriptional program distinct of that of heat shock, which is protective of FUS aggregation. We identified BAG3, a co-chaperone that cooperates with HSP70 in reducing FUS aggregation, as a significant mediator of this effect. We therefore propose BBB-permeable proteasome inhibitors as a potential therapy specific to ALS-FUS.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253043","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 : 2024-09-17DOI: 10.1101/2024.09.17.613482
Rianne van Vredendaal, Léa Joffrin, Antea Paviotti, Claude Mande, Solange Milolo, Nicolas Laurent, Léa Fourchault, Douglas Adroabadrio, Pascal Baelo, Steve Ngoy, Papy Ansobi, Casimir Nebesse, Martine Peeters, Ahidjo Ayouba, Maeliss Champagne, Julie Bouillin, Jana Těšíková, Natalie Van Houtte, Sophie Gryseels, Maha Salloum, Freddy Bikioli, Séverine Thys, Jimmy Mpato, Ruben Ilonga, Henri Kimina, Ynke Larivière, Gwen Lemey, Pierre Van Damme, Jean-Pierre Van Geertruyden, Hypolite Muhindo-Mavoko, Patrick Mitashi, Herwig Leirs, Erik Verheyen, Guy-Crispin Gembu, Joachim Mariën
The wildlife reservoir and spillover mechanisms of the Ebola virus remain elusive despite extensive research efforts in endemic areas. This study employed a One Health approach to examine the virus circulation in wildlife and the associated human exposure risks in the Tshuapa province of the Democratic Republic of the Congo. In 2021, we collected a 1015 samples from 888 animals, predominantly small mammals, and 380 inhabitants of Inkanamongo village, the site of an Ebola virus disease outbreak in 2014. These samples were screened for evidence of current (RNA) or past (IgG antibodies) Ebola virus infections. We also conducted interviews with 167 individuals in the surrounding districts to assess their interactions with wildlife. While no Ebola virus RNA was detected in the wildlife samples, anti-orthoebolavirus IgG antibodies were found in 13 bats and 38 rodents. Among the human participants, 120 individuals had IgG antibodies against at least one orthoebolavirus antigen, with 12 showing seropositivity for two antigens of the same orthoebolavirus, despite not having a prior Ebola disease diagnosis. Furthermore, over 50% of respondents reported frequent visits to the forest to hunt a variety of wild animals, particularly ungulates and rodents, which could account for occasional viral spillovers. The absence of active Ebola virus circulation in wildlife may reflect seasonal patterns in reservoir ecology, like those observed in bats. Similarly, seasonal human activities, like hunting and foraging, may result in periodic exposure risks. These findings highlight the importance of continuous, multidisciplinary surveillance to monitor changes in seasonal spillover risks.
{"title":"Assessing Ebola Virus Circulation in the Tshuapa Province (Democratic Republic of the Congo): A One Health Investigation of Wildlife and Human Interactions","authors":"Rianne van Vredendaal, Léa Joffrin, Antea Paviotti, Claude Mande, Solange Milolo, Nicolas Laurent, Léa Fourchault, Douglas Adroabadrio, Pascal Baelo, Steve Ngoy, Papy Ansobi, Casimir Nebesse, Martine Peeters, Ahidjo Ayouba, Maeliss Champagne, Julie Bouillin, Jana Těšíková, Natalie Van Houtte, Sophie Gryseels, Maha Salloum, Freddy Bikioli, Séverine Thys, Jimmy Mpato, Ruben Ilonga, Henri Kimina, Ynke Larivière, Gwen Lemey, Pierre Van Damme, Jean-Pierre Van Geertruyden, Hypolite Muhindo-Mavoko, Patrick Mitashi, Herwig Leirs, Erik Verheyen, Guy-Crispin Gembu, Joachim Mariën","doi":"10.1101/2024.09.17.613482","DOIUrl":"https://doi.org/10.1101/2024.09.17.613482","url":null,"abstract":"The wildlife reservoir and spillover mechanisms of the Ebola virus remain elusive despite extensive research efforts in endemic areas. This study employed a One Health approach to examine the virus circulation in wildlife and the associated human exposure risks in the Tshuapa province of the Democratic Republic of the Congo. In 2021, we collected a 1015 samples from 888 animals, predominantly small mammals, and 380 inhabitants of Inkanamongo village, the site of an Ebola virus disease outbreak in 2014. These samples were screened for evidence of current (RNA) or past (IgG antibodies) Ebola virus infections. We also conducted interviews with 167 individuals in the surrounding districts to assess their interactions with wildlife. While no Ebola virus RNA was detected in the wildlife samples, anti-orthoebolavirus IgG antibodies were found in 13 bats and 38 rodents. Among the human participants, 120 individuals had IgG antibodies against at least one orthoebolavirus antigen, with 12 showing seropositivity for two antigens of the same orthoebolavirus, despite not having a prior Ebola disease diagnosis. Furthermore, over 50% of respondents reported frequent visits to the forest to hunt a variety of wild animals, particularly ungulates and rodents, which could account for occasional viral spillovers. The absence of active Ebola virus circulation in wildlife may reflect seasonal patterns in reservoir ecology, like those observed in bats. Similarly, seasonal human activities, like hunting and foraging, may result in periodic exposure risks. These findings highlight the importance of continuous, multidisciplinary surveillance to monitor changes in seasonal spillover risks.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"117 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253072","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 : 2024-09-16DOI: 10.1101/2024.09.16.613334
Seyone Chithrananda, Judith Amores, Kevin K Yang
The sense of smell remains poorly understood, especially in contrast to visual and auditory coding. At the core of our sense of smell is the olfactory information flow, in which odorant molecules activate a subset of our olfactory receptors and combinations of unique receptor activations code for unique odors. Understanding this relationship is crucial for unraveling the mysteries of human olfaction and its potential therapeutic applications. Despite this, predicting molecule-OR interactions remains incredibly difficult. Here, we develop a novel, biologically-inspired approach that first maps odorant molecules to their respective OR activation profiles and subsequently predicts their odor percepts. Despite a lack of overlap between molecules with OR activation data and percept annotations, our joint model improves percept prediction by leveraging the OR activation profile of each odorant as auxiliary features in predicting its percepts. We extend this cross receptor-percept approach, showing that sets of molecules with very different structures but similar percepts, a common challenge for chemosensory prediction, have similar predicted OR activation profiles. Lastly, we further probe the odorant-OR model's predictive ability, showing it can distinguish binding patterns across unique OR families, as well as between protein-coding genes or frequently occuring pseudogenes in the human olfactory subgenome. This work may aid in the potential discovery of novel odorant ligands targeting functions of orphan ORs, and in further characterizing the relationship between chemical structures and percepts. In doing so, we hope to advance our understanding of olfactory perception and the design of new odorants with desired perceptual qualities.
人们对嗅觉的了解仍然很少，尤其是与视觉和听觉编码相比。嗅觉的核心是嗅觉信息流，其中气味分子激活嗅觉受体的一个子集，独特的受体激活组合编码出独特的气味。了解这种关系对于揭开人类嗅觉及其潜在治疗应用的神秘面纱至关重要。尽管如此，预测分子与嗅觉受体之间的相互作用仍然非常困难。在这里，我们开发了一种新颖的、受生物学启发的方法，首先将气味分子映射到它们各自的OR激活图谱，然后预测它们的气味感知。尽管分子与受体活化数据和知觉注释之间缺乏重叠，但我们的联合模型利用每种气味的受体活化特征作为预测其知觉的辅助特征，从而改进了知觉预测。我们扩展了这种跨受体-知觉的方法，表明结构迥异但知觉相似的分子集（这是化感预测面临的一个常见挑战）具有相似的预测 OR 激活曲线。最后，我们进一步探究了气味-OR 模型的预测能力，结果表明它可以区分独特的 OR 家族之间的结合模式，以及人类嗅觉亚基因组中的蛋白编码基因或频繁出现的假基因之间的结合模式。这项工作可能有助于发现针对孤儿 OR 功能的新型气味配体，并进一步确定化学结构与感知之间的关系。在此过程中，我们希望加深对嗅觉感知的理解，并设计出具有理想感知品质的新型气味剂。
{"title":"Mapping the combinatorial coding between olfactory receptors and perception with deep learning","authors":"Seyone Chithrananda, Judith Amores, Kevin K Yang","doi":"10.1101/2024.09.16.613334","DOIUrl":"https://doi.org/10.1101/2024.09.16.613334","url":null,"abstract":"The sense of smell remains poorly understood, especially in contrast to visual and auditory coding. At the core of our sense of smell is the olfactory information flow, in which odorant molecules activate a subset of our olfactory receptors and combinations of unique receptor activations code for unique odors. Understanding this relationship is crucial for unraveling the mysteries of human olfaction and its potential therapeutic applications. Despite this, predicting molecule-OR interactions remains incredibly difficult. Here, we develop a novel, biologically-inspired approach that first maps odorant molecules to their respective OR activation profiles and subsequently predicts their odor percepts. Despite a lack of overlap between molecules with OR activation data and percept annotations, our joint model improves percept prediction by leveraging the OR activation profile of each odorant as auxiliary features in predicting its percepts. We extend this cross receptor-percept approach, showing that sets of molecules with very different structures but similar percepts, a common challenge for chemosensory prediction, have similar predicted OR activation profiles. Lastly, we further probe the odorant-OR model's predictive ability, showing it can distinguish binding patterns across unique OR families, as well as between protein-coding genes or frequently occuring pseudogenes in the human olfactory subgenome. This work may aid in the potential discovery of novel odorant ligands targeting functions of orphan ORs, and in further characterizing the relationship between chemical structures and percepts. In doing so, we hope to advance our understanding of olfactory perception and the design of new odorants with desired perceptual qualities.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253046","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 : 2024-09-16DOI: 10.1101/2024.09.16.613350
Randy Aryee, Noor Sakib Mohammed, Supantha Dey, Arunraj B, Swathi Nadendla, Karuna Anna Sajeevan, Matthew Beck, Anthony Nathan Frazier, Jacek Koziel, Thomas Mansell, Ratul Chowdhury
Atmospheric methane (CH4) acts as a key contributor to global warming. As CH4 is a short-lived climate forcer (12 years atmospheric lifespan), its mitigation represents the most promising means to address climate change in the short term. Enteric CH4 (the biosynthesized CH4 from the rumen of ruminants) represents 5.1% of total global greenhouse gas (GHG) emissions, 23% of emissions from agriculture, and 27.2% of global CH4 emissions. Therefore, it is imperative to investigate methanogenesis inhibitors and their underlying modes of action. We hereby elucidate the detailed biophysical and thermodynamic interplay between anti-methanogenic molecules and cofactor F430 of methyl coenzyme M reductase and interpret the stoichiometric ratios and binding affinities of sixteen inhibitor molecules. We leverage this as prior in a graph neural network to first functionally cluster these sixteen known inhibitors among ~54,000 bovine metabolites. We subsequently demonstrate a protocol to identify precursors to and putative inhibitors for methanogenesis, based on Tanimoto chemical similarity and membrane permeability predictions. This work lays the foundation for computational and de novo design of inhibitor molecules that retain/ reject one or more biochemical properties of known inhibitors discussed in this study.
{"title":"Exploring putative enteric methanogenesis inhibitors using molecular simulations and a graph neural network","authors":"Randy Aryee, Noor Sakib Mohammed, Supantha Dey, Arunraj B, Swathi Nadendla, Karuna Anna Sajeevan, Matthew Beck, Anthony Nathan Frazier, Jacek Koziel, Thomas Mansell, Ratul Chowdhury","doi":"10.1101/2024.09.16.613350","DOIUrl":"https://doi.org/10.1101/2024.09.16.613350","url":null,"abstract":"Atmospheric methane (CH4) acts as a key contributor to global warming. As CH4 is a short-lived climate forcer (12 years atmospheric lifespan), its mitigation represents the most promising means to address climate change in the short term. Enteric CH4 (the biosynthesized CH4 from the rumen of ruminants) represents 5.1% of total global greenhouse gas (GHG) emissions, 23% of emissions from agriculture, and 27.2% of global CH4 emissions. Therefore, it is imperative to investigate methanogenesis inhibitors and their underlying modes of action. We hereby elucidate the detailed biophysical and thermodynamic interplay between anti-methanogenic molecules and cofactor F430 of methyl coenzyme M reductase and interpret the stoichiometric ratios and binding affinities of sixteen inhibitor molecules. We leverage this as prior in a graph neural network to first functionally cluster these sixteen known inhibitors among ~54,000 bovine metabolites. We subsequently demonstrate a protocol to identify precursors to and putative inhibitors for methanogenesis, based on Tanimoto chemical similarity and membrane permeability predictions. This work lays the foundation for computational and de novo design of inhibitor molecules that retain/ reject one or more biochemical properties of known inhibitors discussed in this study.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253073","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}
Pannexin 1 (PANX1) forms cell-surface channels capable of releasing signaling metabolites for diverse patho-physiological processes. While inhibiting dysregulated PANX1 is proposed as a therapeutic strategy for many pathological conditions, including inflammatory bowel disease (IBD), low efficacy or poor specificity of classical PANX1 inhibitors introduces uncertainty for their applications in basic and translational research. Here, we performed hit-to-lead optimization and identified a naphthyridone, compound 12, as a new PANX1 inhibitor with an IC50 of 0.73 μM that does not affect pannexin-homologous LRRC8/SWELL1 channels. Using structure-activity relationship analysis, mutagenesis, cell thermal shift assays, and molecular docking, we revealed that compound 12 directly engages PANX1 Trp74 residue. Using a dextran sodium sulfate mouse model of IBD, we found that compound 12 markedly reduced colitis severity, highlighting new PANX1 inhibitors as a proof-of-concept treatment for IBD. These data describe the mechanism of action for a new PANX1 inhibitor, identify the binding site for future drug design, and present a targeted strategy for treating IBD.
{"title":"Novel naphthyridones targeting Pannexin 1 for colitis management","authors":"Wen-Yun Hsueh, Yi-Ling Wu, Meng-Tzu Weng, Shin-Yun Liu, Jascinta P Santavanond, Yi-Chung Liu, Ching-I Lin, Cheng-Nong Lai, Yi-Ru Lu, Jing Yin Hsu, Hong-Yu Gao, Jinq-Chyi Lee, Shu-Chen Wei, Ping-Chiang Lyu, Ivan KH Poon, Hsing-Pang Hsieh, Yu-Hsin Chiu","doi":"10.1101/2024.09.15.613164","DOIUrl":"https://doi.org/10.1101/2024.09.15.613164","url":null,"abstract":"Pannexin 1 (PANX1) forms cell-surface channels capable of releasing signaling metabolites for diverse patho-physiological processes. While inhibiting dysregulated PANX1 is proposed as a therapeutic strategy for many pathological conditions, including inflammatory bowel disease (IBD), low efficacy or poor specificity of classical PANX1 inhibitors introduces uncertainty for their applications in basic and translational research. Here, we performed hit-to-lead optimization and identified a naphthyridone, compound 12, as a new PANX1 inhibitor with an IC50 of 0.73 μM that does not affect pannexin-homologous LRRC8/SWELL1 channels. Using structure-activity relationship analysis, mutagenesis, cell thermal shift assays, and molecular docking, we revealed that compound 12 directly engages PANX1 Trp74 residue. Using a dextran sodium sulfate mouse model of IBD, we found that compound 12 markedly reduced colitis severity, highlighting new PANX1 inhibitors as a proof-of-concept treatment for IBD. These data describe the mechanism of action for a new PANX1 inhibitor, identify the binding site for future drug design, and present a targeted strategy for treating IBD.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"192 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253075","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 : 2024-09-16DOI: 10.1101/2024.09.16.613214
Ivana Borovska, Chundan Zhang, Edoardo Morandi, Daphne van den Homberg, Michael T. Wolfinger, Willem Velema, Danny Incarnato
RNA molecules can populate ensembles of alternative structural conformations, but comprehensively mapping RNA conformational landscapes within living cells presents significant challenges and has, as such, so far remained elusive. Here, we generated the first transcriptome-scale maps of RNA secondary structure ensembles in a living cell, using Escherichia coli as a model. Our analysis uncovered features of structurally-dynamic regions, as well as the existence of hundreds of highly-conserved bacterial RNA structural elements. Conditional structure mapping revealed extensive restructuring of RNA ensembles during cold shock, leading to the discovery of several novel RNA thermometers in the 5′ UTRs of the cspG, cspI, cpxP and lpxP mRNAs. We mechanistically characterized how these thermometers switch structure in response to cold shock and revealed the cspE chaperone-mediated regulation of lpxP. Collectively, this work reveals a previously unappreciated complexity of RNA structural dynamics in living cells, and it provides a key resource to significantly accelerate the discovery of regulatory RNA switches.
{"title":"RNA secondary structure ensemble mapping in a living cell identifies conserved RNA regulatory switches and thermometers","authors":"Ivana Borovska, Chundan Zhang, Edoardo Morandi, Daphne van den Homberg, Michael T. Wolfinger, Willem Velema, Danny Incarnato","doi":"10.1101/2024.09.16.613214","DOIUrl":"https://doi.org/10.1101/2024.09.16.613214","url":null,"abstract":"RNA molecules can populate ensembles of alternative structural conformations, but comprehensively mapping RNA conformational landscapes within living cells presents significant challenges and has, as such, so far remained elusive. Here, we generated the first transcriptome-scale maps of RNA secondary structure ensembles in a living cell, using Escherichia coli as a model. Our analysis uncovered features of structurally-dynamic regions, as well as the existence of hundreds of highly-conserved bacterial RNA structural elements. Conditional structure mapping revealed extensive restructuring of RNA ensembles during cold shock, leading to the discovery of several novel RNA thermometers in the 5′ UTRs of the cspG, cspI, cpxP and lpxP mRNAs. We mechanistically characterized how these thermometers switch structure in response to cold shock and revealed the cspE chaperone-mediated regulation of lpxP. Collectively, this work reveals a previously unappreciated complexity of RNA structural dynamics in living cells, and it provides a key resource to significantly accelerate the discovery of regulatory RNA switches.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"66 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253076","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 : 2024-09-16DOI: 10.1101/2024.09.16.613278
Oluwasegun T Akinniyi, Shardul Kulkarni, Mikayla M Hribal, Cheryl A Keller, Belinda Giardine, Joseph C. Reese
Yeast Def1 mediates RNA polymerase II degradation and transcription elongation during stress. Def1 is predominantly cytoplasmic, and DNA damage signals cause its proteolytic processing, liberating its N-terminus to enter the nucleus. Cytoplasmic functions for this abundant protein have not been identified. Proximity-labeling (BioID) experiments indicate that Def1 binds to an array of proteins involved in posttranscriptional control and translation of mRNAs. Deleting DEF1 reduces both mRNA synthesis and decay rates, indicating transcript buffering in the mutant. Directly tethering Def1 to a reporter mRNA suppressed expression, suggesting that Def1 directly regulates mRNAs. Surprisingly, we found that Def1 interacts with polyribosomes, which requires its ubiquitin-binding domain located in its N-terminus. The binding of Def1 to ribosomes requires the ubiquitylation of eS7a (Rsp7A) in the small subunit by the Not4 protein in the Ccr4-Not complex. Not4 ubiquitylation of the ribosome regulates translation quality control and co-translational mRNA decay. The polyglutamine-rich unstructured C-terminus of Def1 is required for its interaction with decay and translation factors, suggesting that Def1 acts as a ubiquitin-dependent scaffold to link translation status to mRNA decay. Thus, we have identified a novel function for this transcription and DNA damage response factor in posttranscriptional regulation in the cytoplasm and establish Def1 as a master regulator of gene expression, functioning during transcription, mRNA decay, and translation.
{"title":"The DNA damage response and RNA Polymerase II regulator Def1 has posttranscriptional functions in the cytoplasm","authors":"Oluwasegun T Akinniyi, Shardul Kulkarni, Mikayla M Hribal, Cheryl A Keller, Belinda Giardine, Joseph C. Reese","doi":"10.1101/2024.09.16.613278","DOIUrl":"https://doi.org/10.1101/2024.09.16.613278","url":null,"abstract":"Yeast Def1 mediates RNA polymerase II degradation and transcription elongation during stress. Def1 is predominantly cytoplasmic, and DNA damage signals cause its proteolytic processing, liberating its N-terminus to enter the nucleus. Cytoplasmic functions for this abundant protein have not been identified. Proximity-labeling (BioID) experiments indicate that Def1 binds to an array of proteins involved in posttranscriptional control and translation of mRNAs. Deleting DEF1 reduces both mRNA synthesis and decay rates, indicating transcript buffering in the mutant. Directly tethering Def1 to a reporter mRNA suppressed expression, suggesting that Def1 directly regulates mRNAs. Surprisingly, we found that Def1 interacts with polyribosomes, which requires its ubiquitin-binding domain located in its N-terminus. The binding of Def1 to ribosomes requires the ubiquitylation of eS7a (Rsp7A) in the small subunit by the Not4 protein in the Ccr4-Not complex. Not4 ubiquitylation of the ribosome regulates translation quality control and co-translational mRNA decay. The polyglutamine-rich unstructured C-terminus of Def1 is required for its interaction with decay and translation factors, suggesting that Def1 acts as a ubiquitin-dependent scaffold to link translation status to mRNA decay. Thus, we have identified a novel function for this transcription and DNA damage response factor in posttranscriptional regulation in the cytoplasm and establish Def1 as a master regulator of gene expression, functioning during transcription, mRNA decay, and translation.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253363","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 : 2024-09-16DOI: 10.1101/2024.09.16.613242
Harshavardhan Doddapaneni, Sravya V Bhamidipati, Anil Surathu, Hsu Chao, Daniel Paiva Agustinho, Qin Xiang, Kavya Kottapalli, Abirami Santhanam, Zeineen Momin, Kimberly Walker, Vipin K Menon, George M Weissenberger, Nathanael Emerick, Faria Mahjabeen, Qingchang Meng, Jianhong Hu, Richard Sucgang, David Morgan Henke, Fritz Sedlazeck, Ziad Khan, Ginger A Metcalf, Vasanthi Avadhanula, Pedro A Piedra, Sasirekha Ramani, Robert Legare Atmar, Mary K. Estes, Joseph F Petrosino, Richard Gibbs, Donna Muzny, Sara Joan Javornik Cregeen
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples—a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.
{"title":"Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.","authors":"Harshavardhan Doddapaneni, Sravya V Bhamidipati, Anil Surathu, Hsu Chao, Daniel Paiva Agustinho, Qin Xiang, Kavya Kottapalli, Abirami Santhanam, Zeineen Momin, Kimberly Walker, Vipin K Menon, George M Weissenberger, Nathanael Emerick, Faria Mahjabeen, Qingchang Meng, Jianhong Hu, Richard Sucgang, David Morgan Henke, Fritz Sedlazeck, Ziad Khan, Ginger A Metcalf, Vasanthi Avadhanula, Pedro A Piedra, Sasirekha Ramani, Robert Legare Atmar, Mary K. Estes, Joseph F Petrosino, Richard Gibbs, Donna Muzny, Sara Joan Javornik Cregeen","doi":"10.1101/2024.09.16.613242","DOIUrl":"https://doi.org/10.1101/2024.09.16.613242","url":null,"abstract":"Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples—a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253074","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 : 2024-09-15DOI: 10.1101/2024.09.12.612665
Hao Wang, Xiaoqian Xu, Yameng Sun, Hong You, Jidong Jia, You-Wen He, Yuanyuan Kong
Background/Aims: This study aims to determine the predictive capability for metabolic dysfunction-associated steatohepatitis (MASH) long before its diagnosis by using six previously identified diagnostic biomarkers for metabolic dysfunction-associated steatotic liver disease (MASLD) with proteomic data from the UK Biobank. Methods: A nested case-control study comprising of a MASH group and three age- and sex-matched controls groups (metabolic dysfunction-associated steatosis, viral hepatitis, and normal liver controls) were conducted. Olink proteomics, anthropometric and biochemical data at baseline levels were obtained from the UK Biobank. The baseline levels of CDCP1, FABP4, FGF21, GDF15, IL-6 and THBS2 were analyzed prospectively to determine their predictive accuracy for subsequent diagnosis with a mean lag time of over 10 years. Results: At baseline, GDF15 demonstrated the best performance for predicting MASH occurrence at 5 and 10 years later, with an AUC of 0.90 at 5 years and 0.86 at 10 years. A predictive model based on four biomarkers (GDF15, FGF21, IL-6, and THBS2) showed AUCs of 0.88 at both 5 and 10 years. Furthermore, a protein-clinical model that included these four circulating protein biomarkers along with three clinical factors (BMI, ALT and TC) yielded AUCs of 0.92 at 5 years and 0.89 at 10 years. Conclusions: GDF15 at baseline levels outperformed other individual circulating protein biomarkers for the early prediction of MASH. Our data suggest that GDF15 and the GDF15-based model may be used as easy-to-implement tools to identify patients with high risk of developing MASH at a mean lag time of over 10 years.
{"title":"Identification of Growth Differentiation Factor-15 as An Early Predictive Biomarker for Metabolic Dysfunction-Associated Steatohepatitis: A Nested Case-control Study of UK Biobank Proteomic Data","authors":"Hao Wang, Xiaoqian Xu, Yameng Sun, Hong You, Jidong Jia, You-Wen He, Yuanyuan Kong","doi":"10.1101/2024.09.12.612665","DOIUrl":"https://doi.org/10.1101/2024.09.12.612665","url":null,"abstract":"Background/Aims: This study aims to determine the predictive capability for metabolic dysfunction-associated steatohepatitis (MASH) long before its diagnosis by using six previously identified diagnostic biomarkers for metabolic dysfunction-associated steatotic liver disease (MASLD) with proteomic data from the UK Biobank.\u0000Methods: A nested case-control study comprising of a MASH group and three age- and sex-matched controls groups (metabolic dysfunction-associated steatosis, viral hepatitis, and normal liver controls) were conducted. Olink proteomics, anthropometric and biochemical data at baseline levels were obtained from the UK Biobank. The baseline levels of CDCP1, FABP4, FGF21, GDF15, IL-6 and THBS2 were analyzed prospectively to determine their predictive accuracy for subsequent diagnosis with a mean lag time of over 10 years.\u0000Results: At baseline, GDF15 demonstrated the best performance for predicting MASH occurrence at 5 and 10 years later, with an AUC of 0.90 at 5 years and 0.86 at 10 years. A predictive model based on four biomarkers (GDF15, FGF21, IL-6, and THBS2) showed AUCs of 0.88 at both 5 and 10 years. Furthermore, a protein-clinical model that included these four circulating protein biomarkers along with three clinical factors (BMI, ALT and TC) yielded AUCs of 0.92 at 5 years and 0.89 at 10 years. Conclusions: GDF15 at baseline levels outperformed other individual circulating protein biomarkers for the early prediction of MASH. Our data suggest that GDF15 and the GDF15-based model may be used as easy-to-implement tools to identify patients with high risk of developing MASH at a mean lag time of over 10 years.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253413","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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