MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

<div><p>Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.</p></div

Northern blot analysis of yeast total RNAs under acidic conditions.

<p>Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane. The membrane was probed with a mixture of ³²P-labeled oligonucleotides complementary to tRNA Leu (UAA) and tRNA Arg (CCG). Acylated (+) and deacylated (-) tRNAs were loaded on separate lanes. The shift induced by the presence of arginine residues is significantly greater than the one induced by leucine. <i>In vivo</i>, tRNA Leu (UAA) is not significantly arginylated despite a strong affinity for yArgRS.</p

Complexation of yArgRS with isolated tRNAs.

<p>(A) Radiolabeled, <i>in vitro</i> transcribed, tRNA Leu (GAG), Asp (GUC) and Pro (AGG) displaying respectively high, medium and low apparent affinities under competition were tested individually in the presence of various concentrations of yArgRS ranging from 0.25 to 0.50 μM on non-denaturing PAGE. (B) Fractions of complexed tRNA at different enzyme concentrations. The color code corresponds to the relative abundance of each tRNA species at 0.8 μM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130042#pone.0130042.g004" target="_blank">Fig 4</a>, first column).</p

Separation of free and complexed tRNAs by EMSA.

<p>Radiolabeled yeast total tRNAs and in vitro transcribed yeast tRNA Arg (UCU) (as a migration control) were incubated with 0 to 29 μM of yArgRS and separated on a 6% non-denaturing polyacrylamide gel. The fraction of shifted or complexed tRNAs is indicated.</p

Schematic description of the method used for the identification of tRNA complexed by yArgRS.

<p>(A) Yeast total tRNAs were prepared by deacylation and gel purification of a yeast extract of total RNA and subsequently radiolabeled in 5’ with ³²P. (B) Yeast Arginyl-tRNA synthetase (yArgRS) was expressed as a recombinant protein in <i>E</i>. <i>coli</i> and purified by FPLC. (C) Fixed amounts of labeled tRNA were incubated 15 min on ice with varying amounts of purified enzyme ranging from 0.2 to 29 μM. Free tRNAs were separated from complexed species on non-denaturing gel. (D) Complexed tRNAs corresponding to high molecular weight bands were eluted and hybridized on tRNA microarrays.</p

Relative abundance of complexed tRNAs.

<p>Data is shown for four enzyme concentrations all relative to total tRNAs. In other words, it corresponds for each probe, to the relative signal intensity in the complexed tRNA sample divided by the relative signal intensity in total tRNAs (EMSA input), expressed as fold differences. Red indicates an increase in relative amount whereas blue indicates a decrease. Data is organized from high to low relative abundance at 0.8 μM.</p

Yeast tRNA Arg and their decoding capacity.

<p>The figure describes the position on the affinity continuum for yArgRS as well as the arginine codons translated by the four tRNAs, Arg (UCU), (CCG), (ACG) and (CCU). A circle of proportional diameter represents the tRNA gene copy number. tRNA Arg (UCU) and (CCU) both translate AGG codons.</p

<i>Fli1</i>^<i>+/-</i> T cells have significantly lower levels of Neuraminidase 1 (<i>Neu1</i>) message and NEU activity compared to <i>Fli1</i>^<i>+/+</i> T cells during early disease.

<p>cDNA was amplified from RNA isolated from T cells of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old mice (A) and 17-18 week-old mice (C). <i>Neu1</i> and <i>Neu3</i> message levels were measured by real-time PCR and normalized to <i>β-actin</i> levels. B) NEU activity was measured as described in the methods. Relative levels in the NEU activity assay were calculated to combine all animals across experiments as described in the methods. The ‘n’ represents data from individual animals and p values are provided within the figure.</p

Glycosphingolipid levels are significantly reduced in T cells from late disease stage MRL/lpr <i>Fli1</i>^<i>+/-</i> compared to MRL/lpr <i>Fli1</i>^<i>+/+</i> mice.

<p>Glycosphingolipids lactosylceramide (LacCer) (A) and glucosylceramide (GluCer) (B) were measured by SFC/MS/MS in unstimulated or anti-CD3/CD28 stimulated T cells isolated from 17–18 week-old MRL/lpr mice. *p<0.05, **p<0.01, #p<0.005, # #p<0.001. The ‘n’ represents data from individual animals.</p

<i>Fli1</i>^<i>+/-</i> T cells have significantly lower levels of Neuraminidase 1 (<i>Neu1</i>) message and NEU activity compared to <i>Fli1</i>^<i>+/+</i> T cells during early disease.

<p>cDNA was amplified from RNA isolated from T cells of MRL/lpr <i>Fli1</i>^<i>+/+</i> and <i>Fli1</i>^<i>+/-</i> 10-12 week-old mice (A) and 17-18 week-old mice (C). <i>Neu1</i> and <i>Neu3</i> message levels were measured by real-time PCR and normalized to <i>β-actin</i> levels. B) NEU activity was measured as described in the methods. Relative levels in the NEU activity assay were calculated to combine all animals across experiments as described in the methods. The ‘n’ represents data from individual animals and p values are provided within the figure.</p