RNA Modification (eBook)

Chuan He (Herausgeber)

eBook Download: EPUB

2015 | 1. Auflage
408 Seiten
Elsevier Book Series (Verlag)
978-0-12-802331-0 (ISBN)

Infos

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

Textauszug

Chapter One

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: [e-mail]

Abstract

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.

Keywords

Rapid tRNA decay

Poison primer extension

High throughput

Yeast

RNA-ID

SUP4oc

1 Introduction

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 met22Δ 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 trm8Δ trm4Δ 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 trm8Δ trm4Δ 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 trm8Δ trm4Δ 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 trm8Δ trm4Δ 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 trm8Δ trm4Δ 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 tan1Δ trm44Δ mutants (lacking ac4C12 and Um44) and in trm1Δ trm4Δ 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 met22Δ 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 met22Δ 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 met22Δ 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 met22Δ 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...

Produkt-Details

• Erscheint lt. Verlag: 11.12.2015
• Sprache: englisch
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Biochemie / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-802331-7 / 0128023317
• ISBN-13: 978-0-12-802331-0 / 9780128023310