RNA Modification provides a useful examination of the science and its role in biological regulation, the current frontier of life science research, and includes various RNA modications and their role in gene expression. It represents the most up-to-date knowledge and protocols available today.
- Dynamic RNA modifications and their roles in biological regulation are the current frontier of life science research
- This volume of Methods in Enzymology represents up to date knowledge and protocols
1. Methodology For The High-Throughput Identification and Characterization of tRNA Variants that are Substrates for a tRNA Decay Pathway
Matthew J. Payea, Michael P. Guy, and Eric M. Phizicky
2. Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry
Yuriko Sakaguchi, Kenjyo Miyauchi, Byeong-il Kang, and Tsutomu Suzuki
3. A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications
Weiling Maggie Cai, Yok Hian Chionh, Fabian Hia, Chen Gu, Stefanie Kellner, Megan E. McBee, Chee Sheng Ng, Yan Ling Joy Pang, Erin G. Prestwich, Kok Seong Lim, I. Ramesh Babu, Thomas J. Begley and Peter C. Dedon
4. Recognition of Specified RNA Modifications by the Innate Immune System
Tatjana Eigenbrod, Patrick Keller, Steffen Kaiser, Katharina Rimbach, Alexander H. Dalpke and Mark Helm
5. Kinetic Analysis of tRNA Methyl Transferases
Ya-Ming Hou and Isao Masuda
6. Preparation of Human Nuclear RNA m6A Methyltransferases and Demethylases and Biochemical Characterization of Their Catalytic Activity
Jianzhao Liu, Yanan Yue, and Chuan He
7. Transcriptome-Wide Mapping of N6-Methyladenosine by m6A-Seq
Dan Dominissini, Sharon Moshitch Moshkovitz, Ninette Amariglio and Gideon Rechavi
8. Probing RNA Modification Status at Single-Nucleotide Resolution in Total RNA
Nian Liu and Tao Pan
9. High-resolution mapping of N6-methyladenosine in transcriptome and Genome Using a Photo-Crosslinking-Assisted Strategy
Kai Chen, Guan-Zheng Luo and Chuan He
10. Pseudouridine in Mrna: Incorporation, Detection and Recoding
Guowei Wu, Chao Huang and Yi-Tao Yu
11. Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA
T homas M. Carlile, Maria F. Rojas-Duran and Wendy V. Gilbert
12. Pseudouridine Chemical Labeling and Profiling
Xiaoyu Li, Shiqing Ma and Chengqi Yi
12. Experimental Approaches for Target Profiling RNA Cytosine Methyltransferases
Vahid Khoddami, Archana Yerra, and Bradley R. Cairns
13. RNA 5-Methylcytosine Analysis by Bisulfite Sequencing
14. Biochemical and transcriptome-wide identification of A-to-I RNA editing sites by ICE-seq
Shunpei Okada, Masayuki Sakurai, Hiroki Ueda, and Tsutomu Suzuki
15. Radical SAM-Mediated Methylation of Ribosomal RNA
Vanja Stojkovic and Danica Galonic Fujimori
Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway
Matthew J. Payea; Michael P. Guy; Eric M. Phizicky1 Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, New York, USA
1 Corresponding author: email address: firstname.lastname@example.org
The rapid tRNA decay (RTD) pathway is a tRNA quality control pathway known to degrade several specific hypomodified or destabilized tRNAs in the yeast Saccharomyces cerevisiae. In this chapter, we describe seven methods for identifying RTD substrates, with a focus on two new approaches: a high-throughput approach that utilizes a suppressor tRNA library, fluorescence-activated cell sorting, and deep sequencing, and has greatly expanded the known range of RTD substrates; and a poison primer extension assay that allows for the measurement of levels of suppressor tRNA variants, even in the presence of highly similar endogenous tRNAs. We also discuss different applications of the use of the high-throughput and poison primer extension methodologies for different problems in tRNA biology.
Rapid tRNA decay
Poison primer extension
tRNA folding and stability is crucial for efficient translation, and defects in either property can lead to reduced quantities of tRNA, resulting in growth defects in yeast and disease in humans (Hopper, 2013; Yarham, Elson, Blakely, McFarland, & Taylor, 2010). In the yeast Saccharomyces cerevisiae, there are two major cellular quality control pathways known to degrade defective tRNA species. The first pathway is the nuclear surveillance pathway, which acts on pre-tRNA in the nucleus through the use of the nuclear exosome and the TRAMP complex (Kadaba, Wang, & Anderson, 2006; Vanacova et al., 2005) by degrading pre-tRNAiMet lacking the m1A58 modification or with a misprocessed 3´ trailer (Ozanick et al., 2009) and a fraction of wild-type (WT) pre-tRNAs (Gudipati et al., 2012). The second pathway is the rapid tRNA decay (RTD) pathway, which degrades specific mature, hypomodified, or destabilized tRNA species through the activity of the 5´-3´ exonucleases Rat1 and Xrn1 (Alexandrov et al., 2006; Chernyakov, Whipple, Kotelawala, Grayhack, & Phizicky, 2008). RTD is elicited in mutants lacking any of several modifications in the body of the tRNA or through destabilizing mutations, and for all identified RTD substrates, MET22 deletion fully restores tRNA levels and growth (Alexandrov et al., 2006; Chernyakov, Whipple, et al., 2008; Dewe, Whipple, Chernyakov, Jaramillo, & Phizicky, 2012; Guy et al., 2014; Kotelawala, Grayhack, & Phizicky, 2008; Whipple, Lane, Chernyakov, D'Silva, & Phizicky, 2011). Suppression of RTD in met22? strains is presumed to be due to inhibition of the exonucleases Rat1 and Xrn1 by the metabolite 3´-phosphoadenosine-5´-phosphate, which has increased levels when Met22 is inhibited (Dichtl, Stevens, & Tollervey, 1997; Murguia, Belles, & Serrano, 1996).
RTD is known to act on several specific tRNA species, which have been identified and studied using seven approaches (Fig. 1). The first approach was to use microarrays to compare the tRNA levels on a genome-wide scale in trm8? trm4? temperature-sensitive modification mutants (lacking m7G46 and m5C) and in related strains under semipermissive conditions. In this way, we identified the RTD substrate tRNAVal(AAC), since it had reduced tRNA levels in the trm8? trm4? mutant relative to WT or the corresponding single mutants (Alexandrov et al., 2006). Figure 1
Different approaches used to identify and analyze RTD substrates.
In the second approach, northern blots were used to examine both the rate and the specificity of tRNA degradation for RTD substrates in temperature-sensitive modification mutants. In this approach, RNA isolated from cells at different time points after temperature shift was analyzed for levels of specific tRNAs. From this analysis, we found that 50% of the tRNAVal(AAC) was degraded in a trm8? trm4? mutant within 30 min of a shift from 28 to 37 °C, while the similarly hypomodified tRNAiMet, tRNAMet, and tRNAPhe showed no decrease (Alexandrov et al., 2006; Chernyakov, Whipple, et al., 2008). Furthermore, the relative levels of charged and uncharged tRNA could be measured by performing the northern blot under acidic conditions, which showed that levels of charged tRNAVal(AAC) were reduced by 50% within 25 min of temperature shift in a trm8? trm4? mutant and that the uncharged tRNAVal(AAC) levels appeared unaffected (Alexandrov et al., 2006).
The third approach was through high-copy tRNA suppression, wherein a high-copy plasmid expressing a particular tRNA was introduced into a temperature-sensitive tRNA modification mutant. If the tRNA was an RTD substrate and the temperature sensitivity was the result of a single tRNA species being degraded, then overexpression of the tRNA would suppress the defect. Thus, we found that the temperature sensitivity of a trm8? trm4? mutant was suppressed by a high-copy plasmid expressing tRNAVal(AAC), indicating that temperature sensitivity was primarily due to the loss of tRNAVal(AAC), and that the missing modifications were important for tRNA stability (Alexandrov et al., 2006). Similarly, the RTD substrates of several other tRNA modification mutants have also been identified using this approach, including tRNASer(CGA) and tRNASer(UGA) in tan1? trm44? mutants (lacking ac4C12 and Um44) and in trm1? trm4? mutants (lacking m2,2G26 and m5C) (Chernyakov, Whipple, et al., 2008; Dewe et al., 2012; Kotelawala et al., 2008).
Fourth, we used a genetic replacement approach to identify RTD determinants in the tRNASer family by substituting the single essential tRNASer(CGA) gene (SUP61) with different tRNASer(CGA) variants in the WT and the met22? strain, and then assaying for growth at different temperatures. Using this approach, we determined that the combined acceptor and T-stem stabilities were strong determinants for RTD susceptibility in the tRNASer(CGA) gene family (Whipple et al., 2011). This conclusion was further supported by a fifth approach to measure RTD, in which we showed in vitro that tRNASer(CGA) variants lacking ac4C12 and Um44, or with destabilizing mutations in the acceptor stem, were more prone to digestion by Xrn1 and more susceptible to 5´ phosphate removal by calf-intestinal phosphatase (Whipple et al., 2011).
In this review, we will discuss our recently developed sixth and seventh approaches for the study of RTD substrates, which have proven extremely valuable in broadening our understanding of the RTD pathway. The sixth approach uses a fluorescent reporter to comprehensively analyze libraries of thousands of tRNA variants in WT and met22? strains. Through this approach, we have identified 643 likely RTD substrate candidates, many in regions not expected to elicit RTD based on previous work (Guy et al., 2014). We will show data demonstrating that this approach can be used to study tRNA function under different conditions and will discuss other applications of the approach.
The seventh approach employs poison primer extension to measure the tRNA levels in a WT and met22? strain and is valuable in its ability to specifically measure a variant tRNA even in the presence of the WT tRNA whose sequence may differ by only a single residue. We provide a detailed methodology of this approach and discuss some of its other possible applications.
2 High-Throughput Identification of tRNA Substrates Degraded by the RTD Pathway
In this approach, a fluorescent reporter that is sensitive to the levels of functional tRNA is used to identify tRNA variants that are subject to RTD because there is less fluorescence in a WT strain (in which RTD is functional) than in a met22? strain (in which RTD is inhibited). For this analysis, we used the previously developed RNA-ID fluorescent reporter, which contains the inducible PGAL1,10 bidirectional promoter expressing red fluorescent protein (RFP) in one direction and GFPoc (green fluorescent protein (GFP) with a UAA nonsense codon) in the other direction (Dean & Grayhack, 2012). Expression of GFPoc relative to RFP is 0.5% of that of the corresponding GFP reporter without a nonsense codon, and is increased to 94% if the strain has an integrated SUP4oc gene (encoding tRNATyr in which the GUA anticodon is mutated to UUA by a G34U mutation, Fig. 2A), which efficiently suppresses UAA nonsense codons (Guy et al., 2014). We tested the ability of our reporter to distinguish between...