Methods in enzymology最新文献_第6页

Functional analysis of tRNA-derived small translational regulation.

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-03 DOI: 10.1016/bs.mie.2024.11.017

Dongjin Kim, Hak Kyun Kim, Mark A Kay

{"title":"Functional analysis of tRNA-derived small translational regulation.","authors":"Dongjin Kim, Hak Kyun Kim, Mark A Kay","doi":"10.1016/bs.mie.2024.11.017","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.017","url":null,"abstract":"Transfer RNAs (tRNAs) are short non-coding RNA molecules that play a crucial role in protein synthesis by carrying amino acids to ribosomes during translation. tRNAs are highly conserved and abundant across species, with each type categorized based on its anticodon sequence. Although traditionally viewed as essential for protein synthesis, tRNAs have been found to have additional roles in cell proliferation, tumor metastasis, and neuronal homeostasis. In addition, tRNAs are cleaved by ribonucleases to produce smaller fragments. These fragments have previously been referred to as tRNA fragments (tRF RNAs) or tRNA-derived small RNAs (tsRNAs). More recently a nomenclature has been but forward for all tRNA derived RNAs referred to as tDRs. We will use tsRNA and tDR interterchangeably. The tDRs are processed at specific sites in tRNAs and can be differentially expressed in various tissues and diseases, indicating their potential as unique non-coding RNAs with specific functions. In a previous study, we identified a 3'tDR, which can regulate the translation of a target mRNA by altering its secondary structure. This chapter provides a detailed protocol to analyze the tDR-mediated translational regulation based on several molecular methods.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"336-355"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425678","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}

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Preface.

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Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00134-X

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Purification of endogenous tDRs by hybridization-based pulldown.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.019

Yoshika Takenaka, Nana Kunii, Yasutoshi Akiyama

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Detection of mitochondrial tDRs in killifish embryos and other non-model organisms.

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-06 DOI: 10.1016/bs.mie.2024.11.012

Claire L Riggs, Gazal Kalyan, Amie Lt Romney, Jason E Podrabsky

{"title":"Detection of mitochondrial tDRs in killifish embryos and other non-model organisms.","authors":"Claire L Riggs, Gazal Kalyan, Amie Lt Romney, Jason E Podrabsky","doi":"10.1016/bs.mie.2024.11.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.012","url":null,"abstract":"In recent years a diversity of small noncoding RNAs have been identified that originate from the mitochondrial genome. These mitosRNAs are often dominated by tRNA-derived small RNAs (mito-tDRs). Differential expression of mito-tDRs is associated with responses to stress. They also appear to be expressed differentially during development and their expression may be enriched in stress-tolerant animals. Very little is currently known about roles or modes of action of these sequences, although they are implicated in a diversity of processes such as cell cycle regulation, mRNA stability, regulation of ROS production, and import of proteins into the mitochondrion. To better understand the various roles these sequences may play, it is critical that we understand their diversity, cellular location, and the context for their expression. This protocol outlines the methodologies used to detect mitosRNAs, including mito-tDRs, in embryos and cells of the annual killifish Austrofundulus limnaeus. We highlight critical steps in the isolation of RNA, creation of sequencing libraries, bioinformatics processing of sequence data, and methods for validation of expression that support a robust discovery pipeline for mitosRNAs even from species with incomplete reference genome sequences.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"283-311"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425659","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}

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Structural analysis of human ADAR2-RNA complexes by X-ray crystallography.

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.023

Kristen B Campbell, Jeff Cheng, Herra G Mendoza, Agya Karki, Peter A Beal, Andrew J Fisher

{"title":"Structural analysis of human ADAR2-RNA complexes by X-ray crystallography.","authors":"Kristen B Campbell, Jeff Cheng, Herra G Mendoza, Agya Karki, Peter A Beal, Andrew J Fisher","doi":"10.1016/bs.mie.2024.11.023","DOIUrl":"10.1016/bs.mie.2024.11.023","url":null,"abstract":"Adenosine deaminases acting on RNAs (ADARs) are a class of RNA editing enzymes found in metazoa that catalyze the hydrolytic deamination of adenosine to inosine in duplexed RNA. Inosine is a nucleotide that can base pair with cytidine, therefore, inosine is interpreted by cellular processes as guanosine. ADARs are functionally important in RNA recoding events, RNA structure modulation, innate immunity, and can be harnessed for therapeutically-driven base editing to treat genetic disorders. Guide RNAs (gRNAs) bearing various modifications can be used to recruit ADARs to edit sites of interest in a process called site-directed RNA editing (SDRE). To help advance the rational design of gRNAs for therapeutics, characterizing the structure-to-activity relationship of ADARs' recognition and binding of substrate duplex RNA at atomic resolution is critical. In this chapter, we describe the process of determining the structure of human ADAR2 bound to duplex RNA using X-ray crystallography. Solid phase synthesis of 8-azanebularine-modified RNAs and purification for binding and crystallographic studies are described. The overexpression and purification of ADARs and assembly of the protein-RNA complex are detailed. Lastly, methods for crystallizing ADAR-RNA complexes and X-ray structure determination and data refinement strategies are outlined.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"19-53"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11881785/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052994","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

Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis.

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.025

Guoping Li, Saumya Das

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Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis. 直接、集合 FRET 方法监测人类 DNA 聚合酶 δ 全酶组装和 DNA 合成启动的瞬态动力学。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-28 DOI: 10.1016/bs.mie.2024.08.002

Jessica L Norris, Mark Hedglin

{"title":"Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.","authors":"Jessica L Norris, Mark Hedglin","doi":"10.1016/bs.mie.2024.08.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.08.002","url":null,"abstract":"In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"271-309"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400652","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

PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step. PAR-dCLIP：通过加入解帽步骤，检测结合在 5'末端的 RNA 结合蛋白目标转录本。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-07 DOI: 10.1016/bs.mie.2024.08.003

Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano

{"title":"PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step.","authors":"Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano","doi":"10.1016/bs.mie.2024.08.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.08.003","url":null,"abstract":"RNA binding proteins (RBPs) are responsible for facilitating a wealth of post-transcriptional gene regulatory functions. The role of an RBP on regulated transcripts can be investigated through a pull-down of the RBP and high-throughput sequencing (HTS) of the associated transcripts. Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), is one such pull-down method that isolates, detects, and sequences the cDNA of RBP-associated transcripts. PAR-CLIP relies on a photoactivatable ribonucleoside analogue, 4-thiouridine, to facilitate covalent RNA-protein crosslinks at 365 nm. These crosslinks permit stringent wash conditions and result in T to C mismatch incorporations during reverse transcription, a unique parameter for the computational analysis of high-confidence binding sites. However, until now, RBPs that bind at the 5'-termini of RNAs have been uniquely restricted from the full potential bandwidth of autoradiographic detection and HTS library preparation. The 5'-termini of RNAs are highly modified, including the most common Pol-II derived modification: the 7-methylguanosine (m7G) cap. In the conventional PAR-CLIP protocol, cap-binding proteins protect the m7G cap from the RNase treatment that generates the necessary substrate for autoradiographic detection and 5' adapter ligation-thus occluding entire populations of RNA from visualization and HTS. Here, we introduce decapping-PAR-CLIP or PAR-dCLIP. We incorporate a decapping step into the PAR-CLIP protocol to generate the necessary substrate to sequence m7G capped transcripts. While PAR-dCLIP was originally targeted towards known m7G-cap binding proteins, we argue that all RBP inquiries, and particularly those suspected to regulate translation, should incorporate this decapping step to ensure that all possible populations of bound transcripts are identified.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"159-222"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400658","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

In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01. 荧光假单胞菌 IP01 的三组分 Rieske 加氧酶--积烯二加氧酶的体外分析。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-12 DOI: 10.1016/bs.mie.2024.05.013

Niels A W de Kok, Hui Miao, Sandy Schmidt

{"title":"In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.","authors":"Niels A W de Kok, Hui Miao, Sandy Schmidt","doi":"10.1016/bs.mie.2024.05.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.013","url":null,"abstract":"Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"167-192"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291291","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

Purification of functional recombinant human mitochondrial Hsp60. 纯化功能性重组人线粒体 Hsp60。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mie.2024.07.049

Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem

{"title":"Purification of functional recombinant human mitochondrial Hsp60.","authors":"Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem","doi":"10.1016/bs.mie.2024.07.049","DOIUrl":"10.1016/bs.mie.2024.07.049","url":null,"abstract":"The mitochondrial 60 kDa heat shock protein (mHsp60) is an oligomeric, barrel-like structure that mediates protein folding in cooperation with its cochaperonin Hsp10, in an ATP-dependent manner. In contrast to the extremely stable oligomeric structure of the bacterial chaperonin, GroEL, the human mHsp60 exists in equilibrium between single and double heptameric units, which dissociate easily to inactive monomers under laboratory conditions. Consequently, purification and manipulation of active mHsp60 oligomers is not straightforward. In this manuscript, we present an improved protocol for the purification of functional mHsp60, following its expression in bacteria. This method is based upon a previously published strategy that exploits the notorious instability of mHsp60 to purify the monomeric form, which is subsequently reconstituted to functional oligomers under controlled conditions. In our protocol, we use affinity chromatography on a Ni NTA-agarose resin as the initial step, facilitating purification of substantial amounts of highly pure active protein. The resulting Hsp60 is suitable for both functional and structural analyses, including crystallography and electron cryo-microscopy (cryo-EM) studies, to obtain high resolution structures of the mHsp60 oligomers alone and in various complexes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"423-440"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564646","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