A robust method for measuring aminoacylation through tRNA-Seq

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge transfer RNA sequencing (tRNA-Seq) method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.

Figure 1-gure supplement 1.Schematic of the Whitfeld reaction with acylated and deacylated tRNA leading to generation of CCA and CC-ending tRNAs.For deacylated tRNA, 3' adenosine is oxidized by periodate and then cleaved o by lysine induced -elimination (Rammler, 1971;Uziel, 1973).Acylated tRNA is protected from periodate oxidation but will be deacylated in the subsequent incubation with lysine.(A) Aminoacylation remaining after 5, 30, 90 and 270 min of deacylation in 1 M lysine pH=8 at 45°C.After deacylation, RNA was puri ed and submitted to the Whitfeld reaction using lysine cleavage at pH=9.5 for 90 min at 45°C to ensure complete deacylation.The RNA was then processed using the described charge tRNA-Seq method.(B) RNA stability over time for lysine cleavage at pH=8 and borax cleavage at pH=9.5.(C) Lysine reacts with dialdehydes forming from quencher oxidation.One-pot Whitfeld reactions were performed at pH=8 and pH=9.5 and quenched with either water (MQ), ethylene glycol (Egl), glycerol (Gly), glucose (Glc) or ribose (Rib).Pictures taken before (0 h) and after (4 h) the lysine cleavage step indicate side product formation consistent with lysine reacting with dialdehydes formed during the periodate quenching (Saraiva et al., 2006).This side product causes problems in the later puri cation step.For density plots, a negative deviation is the result of RPM or charge readings being higher for TGIRT than for Maxima, and vice versa for positive deviations.

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Figure 2-gure supplement 1. Optimizing lysine induced cleavage for the charge tRNA-Seq method.(A)Aminoacylation remaining after 5, 30, 90 and 270 min of deacylation in 1 M lysine pH=8 at 45°C.After deacylation, RNA was puri ed and submitted to the Whitfeld reaction using lysine cleavage at pH=9.5 for 90 min at 45°C to ensure complete deacylation.The RNA was then processed using the described charge tRNA-Seq method.(B) RNA stability over time for lysine cleavage at pH=8 and borax cleavage at pH=9.5.(C) Lysine reacts with dialdehydes forming from quencher oxidation.One-pot Whitfeld reactions were performed at pH=8 and pH=9.5 and quenched with either water (MQ), ethylene glycol (Egl), glycerol (Gly), glucose (Glc) or ribose (Rib).Pictures taken before (0 h) and after (4 h) the lysine cleavage step indicate side product formation consistent with lysine reacting with dialdehydes formed during the periodate quenching(Saraiva et al., 2006).This side product causes problems in the later puri cation step.

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Figure 2-gure supplement 2. Measurement bias in charge tRNA-Seq using blunt-end ligation.(A) Measured charge of a E.coli tRNA-Lys oligo control spiked into samples processed with four di erent pre-adenylated adapters.The control was made using a mix of 50% E.coli tRNA-Lys-CCA and 50% E.coli tRNA-Lys-CC and thus simulating 50% charge.Each dot represents a single charge tRNA-Seq sample.(B) Distribution of charge di erences at the transcript level among samples with two barcode replicates, comparing adapters A1 vs. A2, A2 vs. A3 and A3 vs. A4.Deviation is reported as percentage point di erences and the kernel density estimate (KDE) is overlaid.

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Figure 2-gure supplement 5. (A) Ligation test comparing the e ect of RNA processing.Similar to Figure 2, panel E but with two di erent adapters.(B)The unligated tRNA that appears after tRNA is oxidized with periodate in panel A is refractory to further ligation.The unligated tRNA was gel puri ed from enough ligation reactions as shown in panel A to setup two new ligation reactions using either l1N pre-adenylated adapter for blunt end ligation or l6Sp for splint assisted ligation.For l1N, ligation was setup with 35 ng tRNA, 20 pmol adapter, 17.5% PEG-8000, 20% DMSO, 1xT4 RNA ligase bu er, 1 L T4 RNA ligase 2 (truncated KQ) and 1 L SuperaseIn.For l6Sp, the ligation was setup as described in the charge tRNA-Seq protocol.

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Figure2-gure supplement 8. Charge tRNA-Seq control samples and spike-ins validate the method.(A) Cleavage of the 3' adenosine on spike-in oligo is near complete and similarly measured across adapters.Using the E.coli tRNA-Lys-CCA oligo as a spike-in control to monitor completion of the Whitfeld reaction.If complete, 100% E.coli tRNA-Lys-CC should be produced and thus appearing as 0% charged.Each dot represents one sample spiked with E.coli tRNA-Lys-CCA oligo before the Whitfeld reaction and processed using the charge tRNA-Seq processing described in the method section.(B) Aminoacylation level of human tRNA transcripts after undergoing deacylation by incubation at 45°C for 4 h in 1 M lysine (pH=8).Mitochondrial tRNA fMet was excluded because formylated amino acids are known to be highly resistant towards deacylation (Scho eld and Zamecnik, 1968).(C) Aminoacylation level of tRNA transcripts from four samples receiving sham oxidation (NaCl) during the Whitfeld reaction.

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Figure 2-gure supplement 9. Charge measurement comparison between H1299 at baseline (this work) and 293T at baseline (Evans et al., 2017).Each point is an isodecoder shared by both datasets, errorbars are plus/minus one standard deviation of the sample replicates and the red line is equality.H1299 charge measurements with four replicates also displayed in Figure 2, panel F. 293T charge measurement with six replicates, from NCBI Geo database (accession GSE97259, supplementary le).In right text box, the average charge and the average replicate standard deviation, across all isodecoders.

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Figure 3-gure supplement 3. Polymerase dependent mismatch frequency, gap frequency and RT stop percentage for the mcm 5 s 2 U modi ed position.Comparing TGIRT vs. Maxima RT polymerases, otherwise similar to Figure 3-gure Supplement 2. Data is shown for 7 individual RNA samples that were submitted to the Whitfeld reaction, barcoded, pooled and used for RT-PCR template with both TGIRT-III and Maxima.

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Figure 4-gure supplement 1. Best and worst pairwise comparisons between barcode replicates.Sorting pairwise comparisons between barcode replicates according to the sum of squared di erences and showing the best and worst either at the transcript or codon level.(A) For charge levels, adapter l4Sp tends to overestimate charge.(B) For RPM levels.For all plots the red line is equality.

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Figure 5-gure supplement 2. Charge titration prediction error binned by sequencing run and titration sample.(A) Run-to-run bias of two sequencing libraries independently prepared and sequenced on di erent days.(B) Error distribution binned by titration sample.In both panels, error is the percentage point di erence between the measured vs. predicted charge for all transcripts in the bin.