Methods in enzymology最新文献

Northern blotting (NB) is a classic method for visualizing the length as well as the amount of specific RNA using gel separation and hybridization probes. As transfer RNA-derived RNAs (tDRs) are generated from mature tRNAs or pre-tRNAs, the ratio of tDR to mature tRNA or pre-tRNA will be a useful information about the efficiency of tDR production. By designing NB probes which hybridize to a mature tRNA of interest, the blot can simultaneously visualize the amount of tDRs as well as mature tRNAs and pre-tRNAs originated from the same gene, which is a significant advantage of NB. In this chapter, we present a protocol for the detection of tDRs or pre-tRNAs by NB using denaturing polyacrylamide gel electrophoresis and Digoxigenin-dUTP-tailed oligo DNA probes. Through example experiments, we show that tDRs originating from the same mature tRNA can be differentiated based on their length. We also show that our method can be applied to the evaluation of pre-tRNA processing.

Transfer RNA (tRNA)-derived small RNAs (tDRs) are emerging as a novel class of regulatory molecules with significant implications in gene expression and cellular processes. These tDRs are generated through precise cleavage of precursor or mature tRNAs and can function in a sequence dependent manner or structure dependent manner. Recent studies have uncovered a unique subset of tDRs that can form tetramolecular assemblies, adding a new layer of complexity to their functional repertoire. Tetramolecular tDRs exhibit remarkable stability and functional diversity, influencing processes such as translation regulation, stress response, and cellular signaling. The assembly of these tDRs into tetramers is facilitated by guanine-rich sequence motifs which promote intermolecular interactions essential for their structure and biological activity. Understanding the formation, structural dynamics, and functional roles of tetramolecular tDRs offers new insights into tDR-mediated gene regulation and the potential development of RNA-based therapeutic strategies. This article aims to discuss a set of biochemical, biophysical, and reporter assay-based techniques that can be used to characterize G-quadruplex structures formed by tDRs.

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.

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.

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.

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.

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.

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.

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.