Methods in enzymology最新文献

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.

As transfer RNAs (tRNAs) are characterized by the existence of a variety of post-transcriptional modifications, transfer RNA-derived RNAs (tDRs) also possess various modifications. Accumulating evidence suggests that these modifications can regulate the biogenesis and the biological functions of tDRs. Therefore, it is important to purify endogenous tDRs for examining the physiological roles of tDRs. Here we present a simple protocol for purification of endogenous tDRs by hybridization-based pulldown. In this method, tDRs of interest are hybridized to biotinylated oligo DNA probes, followed by pulldown using a streptavidin agarose resin. Resin-bound tDR-probe complexes are then isolated by competitive dissociation using excess amount of biotin. After digestion of probes by DNase I, the purified tDRs are obtained. As the pulldown efficiency of this method largely depends on how efficiently tDRs are generated, the yield can be significantly improved by combination with methods for efficient tDR production, such as in lysate RNA digestion method that we previously reported.

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5'- and 3'- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

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.

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.

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.

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.

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.

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.