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Analyzing, visualizing, and annotating tRNA-derived RNAs using tRAX and tDRnamer.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mie.2024.11.016
Patricia P Chan, Andrew D Holmes, Todd M Lowe
{"title":"Analyzing, visualizing, and annotating tRNA-derived RNAs using tRAX and tDRnamer.","authors":"Patricia P Chan, Andrew D Holmes, Todd M Lowe","doi":"10.1016/bs.mie.2024.11.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.016","url":null,"abstract":"<p><p>tRNA-derived RNAs (tDRs) are known for their diverse regulatory roles in many organisms. These small RNA transcripts have been identified mainly by high-throughput RNA sequencing, numbering hundreds to thousands of unique molecules in any given biological sample. As such, bioinformatic analysis is essential in understanding the features, complexity, and unexplored biological patterns of tDRs. This chapter describes use of tRAX: tRNA Analysis of eXpression, a specially designed comprehensive end-to-end software pipeline for tDR abundance estimation, differential expression comparison, and inference of RNA modifications from raw small RNA sequencing data. We also demonstrate tDRnamer, a web- and command-line-based companion tool that provides automated, standardized tDR naming and annotations based on source tRNAs and related tDRs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"103-133"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425703","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A general framework to over-express tRNA-derived fragments from their parental tRNAs in mammalian cells.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.008
Fatemeh Esmaeili, Kumarjeet Banerjee, Zhangli Su, Anindya Dutta
{"title":"A general framework to over-express tRNA-derived fragments from their parental tRNAs in mammalian cells.","authors":"Fatemeh Esmaeili, Kumarjeet Banerjee, Zhangli Su, Anindya Dutta","doi":"10.1016/bs.mie.2024.11.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.008","url":null,"abstract":"<p><p>tRNA-derived fragments (tRFs), generated from the cleavage of mature or precursor tRNAs are a category of regulatory noncoding RNAs with diverse functions in physiological or pathophysiological conditions. Here we describe a framework for the over-expression of tRFs from their parental tRNAs in mammalian cells. The process involves bioinformatics analysis to identify specific tRNAs that produce the tRF, PCR amplification of corresponding tRNA genes, and insertion into expression vectors. Transfection is carried out in HEK293T cells and detection of tRFs is achieved through northern blotting and dual luciferase reporter assays. In the latter, a complementary sequence to the tRF of interest is inserted into the luciferase reporter. By observing the reduction in luciferase activity, we can validate the expression of tRFs. This method enables precise study of tRF functions and their roles in cellular processes.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"241-259"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Experimental strategy for characterization of novel TnpB orthologs.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-05 DOI: 10.1016/bs.mie.2025.01.056
Gytis Druteika, Tautvydas Karvelis, Virginijus Šikšnys
{"title":"Experimental strategy for characterization of novel TnpB orthologs.","authors":"Gytis Druteika, Tautvydas Karvelis, Virginijus Šikšnys","doi":"10.1016/bs.mie.2025.01.056","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.056","url":null,"abstract":"<p><p>TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"183-195"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692773","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Discrimination between vesicular and nonvesicular extracellular tRNAs and their fragments.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.042
Mauricio Castellano, Marco Li Calzi, Maria Rosa Garcia, Alfonso Cayota, Juan Pablo Tosar
{"title":"Discrimination between vesicular and nonvesicular extracellular tRNAs and their fragments.","authors":"Mauricio Castellano, Marco Li Calzi, Maria Rosa Garcia, Alfonso Cayota, Juan Pablo Tosar","doi":"10.1016/bs.mie.2024.11.042","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.042","url":null,"abstract":"<p><p>The extracellular space contains RNAs both inside and outside extracellular vesicles (EVs). Among RNA types, tRNAs and tRNA-derived small RNAs (tDRs) tend to be abundant and are frequently detected when performing small RNA sequencing of extracellular samples. For several applications, including answering basic biology questions and biomarker discovery, it is important to understand which specific extracellular tRNAs and tDRs are inside EVs and which are not. We have observed that EVs contain mainly full-length tRNAs, while cells also release full-length tRNAs into nonvesicular fractions. However, these nonvesicular tRNAs are fragmented by extracellular ribonucleases into nicked tRNAs, which can dissociate into tDRs both in extracellular samples and in the laboratory. It is therefore crucial to separate EVs from other nonvesicular RNA-containing extracellular carriers to prevent cross-contamination. Otherwise, extracellular tDR profiling may mix up signals coming from structurally and functionally different carrier types. Here, we provide two protocols that achieve this by: (a) density gradient separation and, (b) the use of commercial, pre-packed size-exclusion chromatography columns. The first protocol is time-consuming but achieves high resolution, while the second protocol is faster, simpler, and recommended for routine separations. Taken together, they form a solid experimental toolkit for addressing different questions related to extracellular tRNA biology or biomarker discovery.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"171-185"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Ribosome-associated tDRs in yeast.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.006
Alessia Rosina, Norbert Polacek, Robert Rauscher
{"title":"Ribosome-associated tDRs in yeast.","authors":"Alessia Rosina, Norbert Polacek, Robert Rauscher","doi":"10.1016/bs.mie.2024.11.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.006","url":null,"abstract":"<p><p>The regulation of gene expression in response to environmental stress is a key process that ensures cellular survival across all three domains of life. The adjustment of protein synthesis appears to be one of the initial steps toward the response and adaptation to stress. Ribosome-associated non-coding RNAs (rancRNAs) efficiently regulate translation as an immediate response to stress by directly targeting the ribosome and fine-tuning translation. tRNA-derived RNAs (tDRs) are part of the RNA species that constitute the functionally diverse class of rancRNAs. Here we report a new experimental approach for creating deep sequencing libraries of ribosome-associated small RNAs in yeast utilizing state-of-the-art technologies. Our new strategy is supported by validating previously identified rancRNAs and discovering novel tDRs interacting with the Saccharomyces cerevisiae ribosome.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"85-101"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-19 DOI: 10.1016/bs.mie.2025.01.052
Satoshi N Omura, Osamu Nureki
{"title":"General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.","authors":"Satoshi N Omura, Osamu Nureki","doi":"10.1016/bs.mie.2025.01.052","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.052","url":null,"abstract":"<p><p>Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"23-39"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Genome editing with programmable base editors in human cells.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-05 DOI: 10.1016/bs.mie.2025.01.001
Nicola R B Osgood, Natalie M Zawalick, Courtney B Sawyer, Quinn T Cowan, Sifeng Gu, S J Mawson, Brodie L Ranzau, Lehan Li, Melissa Gymrek, Alon Goren, Alexis C Komor
{"title":"Genome editing with programmable base editors in human cells.","authors":"Nicola R B Osgood, Natalie M Zawalick, Courtney B Sawyer, Quinn T Cowan, Sifeng Gu, S J Mawson, Brodie L Ranzau, Lehan Li, Melissa Gymrek, Alon Goren, Alexis C Komor","doi":"10.1016/bs.mie.2025.01.001","DOIUrl":"10.1016/bs.mie.2025.01.001","url":null,"abstract":"<p><p>Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"351-404"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11948366/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bioinformatic approaches for accurate assessment of A-to-I editing in complete transcriptomes.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-23 DOI: 10.1016/bs.mie.2024.11.020
Eli Eisenberg
{"title":"Bioinformatic approaches for accurate assessment of A-to-I editing in complete transcriptomes.","authors":"Eli Eisenberg","doi":"10.1016/bs.mie.2024.11.020","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.020","url":null,"abstract":"<p><p>A-to-I RNA editing is an RNA modification that alters the RNA sequence relative to the its genomic blueprint. It is catalyzed by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes, and contributes to the complexity and diversification of the proteome. Advancement in the study of A-to-I RNA editing has been facilitated by computational approaches for accurate mapping and quantification of A-to-I RNA editing based on sequencing data. In this chapter we review some of the main computational approaches currently used, describe potential hurdles, challenges and pitfalls, and discuss possible ways to mitigate them.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"241-265"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Leveraging Saccharomyces cerevisiae for ADAR research: From high-yield purification to high-throughput screening and therapeutic applications.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.11.026
Adi Akira, Erez Levanon, Shay Ben Aroya
{"title":"Leveraging Saccharomyces cerevisiae for ADAR research: From high-yield purification to high-throughput screening and therapeutic applications.","authors":"Adi Akira, Erez Levanon, Shay Ben Aroya","doi":"10.1016/bs.mie.2024.11.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.026","url":null,"abstract":"<p><p>Saccharomyces cerevisiae, a model eukaryotic organism with a rich history in research and industry, has become a pivotal tool for studying Adenosine Deaminase Acting on RNA (ADAR) enzymes despite lacking these enzymes endogenously. This chapter reviews the diverse methodologies harnessed using yeast to elucidate ADAR structure and function, emphasizing its role in advancing our understanding of RNA editing. Initially, Saccharomyces cerevisiae was instrumental in the high-yield purification of ADARs, addressing challenges associated with enzyme stability and activity in other systems. The chapter highlights the successful application of yeast in high-throughput screening platforms that identify key structural motifs and substrate preferences of ADARs, showcasing its utility in revealing complex enzyme mechanics. Furthermore, we discuss the development of yeast-based systems to optimize guide RNA sequences for site-directed RNA editing (SDRE), demonstrating how these systems can be employed to refine therapeutic strategies targeting genetic mutations. Additionally, exogenous expression of ADARs from various species in yeast has shed light on enzyme potency and substrate recognition across different temperatures, offering insights into evolutionary adaptations. Overall, Saccharomyces cerevisiae has proven to be an invaluable asset in ADAR research, facilitating significant advances in our understanding of RNA editing mechanisms and therapeutic applications.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"1-18"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.005
Vera Oberbauer, Aleksej Drino, Matthias R Schaefer
{"title":"Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.","authors":"Vera Oberbauer, Aleksej Drino, Matthias R Schaefer","doi":"10.1016/bs.mie.2024.11.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.005","url":null,"abstract":"<p><p>tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"356-380"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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