Methods in enzymology最新文献

RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.

tRNA fragments (tRFs) are generated by cellular endogenous ribonuclease cleavage and play important roles in cellular processes and diseases states. Many questions regarding tRF functions remain to be studied and understood. Common sequencing techniques measure tRF after a size selection step that separates the full-length tRNA and tRF before sequencing library construction. The crucial information on the relationship of tRFs to their respective full-length tRNA in the same biological sample cannot be obtained in this way. We developed multiplex small RNA sequencing (MSR-seq) which measures the abundance as well as site-specific modification information on both full-length tRNA and their matching tRFs in the same sample. Here we describe the bioinformatic steps to obtain the tRF abundance data from the MSR-seq data using the publicly available pipeline in Github (https://github.com/Luke-F1875/MSRseq_data_processing_pipeline).

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

Although RNA-seq data are becoming more widely available for biomedical research, most datasets for short non-coding RNAs (sncRNAs) primarily focus on microRNA analysis using standard RNA-seq, which captures only sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. Standard RNA-seq fails to sequence sncRNAs with different terminal phosphate states, including tRNA halves, the most abundant class of tRNA-derived sncRNAs that play diverse roles in various biological processes. tRNA halves are produced through the endoribonucleolytic cleavage of mature tRNA anticodon loops. The responsible endoribonucleases, such as Angiogenin, commonly leave a 2',3'-cyclic phosphate (cP) at the 3'-end of 5'-tRNA halves and forms a 5'-OH end of 3'-tRNA halves, making them incompatible with standard RNA-seq. We developed a method named "cP-RNA-seq" that selectively amplifies and sequences tRNA halves and other cP-containing sncRNAs. Here we describe a detailed and recently updated cP-RNA-seq protocol. In this method, the 3'-end of all sncRNAs, except those containing a cP, are cleaved through periodate treatment after phosphatase treatment. Consequently, adaptor ligation and cDNA amplification steps are exclusively applied to cP-containing sncRNAs. Our cP-RNA-seq only requires commercially available reagents and is broadly applicable for the global identification of tRNA halves and other cP-containing sncRNA repertoires in various transcriptomes.

Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Site-directed RNA editing (SDRE) holds significant promise for treating genetic disorders resulting from point mutations. Gene therapy, for common genetic illnesses is becoming more popular and, although viable treatments for genetic disorders are scarce, stop codon mutation-related conditions may benefit from gene editing. Effective SDRE generally depends on introducing many guideRNA molecules relative to the target gene; however, large ratios cannot be achieved in the context of gene therapy applications. Gene-encoded information can be altered, and functionally diverse proteins produced from a single gene by restoration of point-mutated RNA molecules using SDRE. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme, that can specifically convert adenosine (A) residues to inosines (I), which are translated as guanosine (G). MS2 system along with ADAR1 deaminase domain can target a particular A and repair G to A mutations. In this study, we used the RNA binding MS2 coat protein fused with the ADAR1 deaminase domain controlled by the CMV promoter, and a 19 bp guide RNA (complementary to the target mRNA sequence) engineered with 6 × MS2 stem-loops downstream or 1 × MS2 stem-loop (double MS2) on either side, controlled by the U6 promoter. When the EGFP TGG codon (tryptophan) was altered to an amber (TAG), opal (TGA), or ochre (TAA) stop codon, the modified ADAR1 deaminase domain could convert A-to-I (G) at the edited sites. It is anticipated that successful establishment of this technique will result in a new era in gene therapy, allowing remarkably efficient gene repair, even in vivo.

Adenosine to inosine deaminases acting on RNA (ADARs) enzymes are found in all metazoa. Their sequence and protein organization is conserved but also shows distinct differences. Moreover, the number of ADAR genes differs between organisms, ranging from one in flies to three in mammals. The distinct isoforms of ADARs and their specific roles determine the complexity of A-to-I RNA editing, its regulation and the versatility of these enzymes. Understanding the different isoform-specific functions and targets will provide a deeper understanding of the diverse biological processes influenced by ADARs, either through ADAR editing of dsRNAs or the interaction with RNAs and proteins. The detailed identification and assigning of isoform-specific targets is a crucial step towards our understanding of functional differences amongst ADAR isoforms and will help us to understand their individual implications for health and disease. This chapter delves into unique characteristics and functional implications of ADAR isoforms. We describe the ectopic overexpression in editing free cells and the use of RNA immunoprecipitation coupled with sequencing to determine isoform-specific interactions with RNAs and their editing sites. Additionally, we discuss new challenges in editing detection by different ADARs in the context of other modifications and provide ideas for potentially better methods to determine the "true editome".

When quantifying tRNA-derived short non-coding RNAs (sncRNAs), two key considerations must be addressed. First, sequencing analyses have revealed significant heterogeneity in the lengths and terminal sequences of tRNA-derived sncRNAs. Second, within the total RNA fraction, these sncRNAs coexist with more abundant mature tRNAs and their precursors (pre-tRNAs), which share identical sequences with the sncRNAs. While accurate quantification of individual tRNA-derived sncRNAs is crucial for research on these molecules, these two factors make it challenging to achieve with standard RT-qPCR, stem-loop RT-qPCR, and northern blot. We have developed a TaqMan RT-qPCR method that specifically quantifies tRNA half molecules. Here we describe a detailed and recently updated protocol in which an adaptor is ligated to the target tRNA half, and the TaqMan probe targets the boundaries of the tRNA half and adaptor, ensuring specific quantification without cross-reacting with corresponding mature tRNA or pre-tRNA. Our method utilizes only commercially available reagents and is broadly applicable for quantifying tRNA halves and other sncRNAs in diverse samples, including clinical specimens such as human plasma.