| Summary | An integrated method that uses chemical ligation to sandwich the amino acid of a charged tRNA in between the body of the tRNA and an adaptor oligonucleotide, followed by high throughput nanopore sequencing. |
| Key findings | Loss of modifications by TRM4 and TRM8 leads to destabilization and de-aminoacylation of tRNA-Val-AAC |
| Sequencing strategy | ONT dorect RNA sequencing |
| Raw data | https://www.ebi.ac.uk/ena/browser/view/PRJEB90828 |
| Software/data repository | https://github.com/rnabioco/aa-tRNA-seq; https://doi.org/10.5281/zenodo.15653411 |
| Experimental protocol | Chemical ligation. The ligation reactions were set up by diluting the aminoacylation reactions eightfold so that the final solution contained 5 µM of the aminoacylated tRNA, 50 µM 5′-phosphorimidazolide adapter, 50 µM splint (also called the 5′-adapter below), 5 mM EDTA, and 37.5 mM HEPES pH 8. The reactions were allowed to proceed for 24 h on ice, before being diluted with an equal volume of a solution of 5 mM EDTA, 99% v/v formamide and purified by 16% denaturing PAGE. The ligated products were cut out from the gel, crushed, and soaked in a solution of 2 mM EDTA, 5 mM sodium acetate acidified to pH 5 on a rotator for 3 h at 4 °C. The extracted aa-bridged tRNA products were then filtered using 0.22 µm spin filters, concentrated using Amicon 10 k MWCO filters, and desalted using the Oligo Clean and Concentrator kit (Zymo Research). After chemical ligation and gel purification, the Cys-bridged tRNA was reduced with DTT for 1 h at room temperature. The reaction contained 1.2 µM Cys-bridged tRNA, 50 mM HEPES pH 8, and 10 mM DTT. After the 1 h incubation, the reduction reaction was diluted 1.33-fold so that the final alkylation solution contained 0.9 µM Cys-bridged tRNA, 37.5 mM HEPES pH 8, 7.5 mM DTT, and 50 mM chloroacetamide. The alkylation reaction was allowed to proceed for 30 min in the dark, after which it was cleaned up using the RNAClean XP beads (Beckman Coulter) according to the manufacturer protocol with the following change: immediately after the addition of the bead suspension to the ligation reaction, isopropanol equal to volume of the reaction+beads was added. Yeast pellets were thawed on ice and resuspended in 400 µL of cold AES (10 mM NaOAc pH 4.5, 1 mM ETDA pH 8, 0.5% SDS). 400 µL of cold 25:24:1 acid phenol:chloroform:isoamyl alcohol was added. Samples were vortexed for 15 s and allowed to rest on ice for 20 min, vortexing every 5 min. They were then spun at 18,000 × g for 10 min at 40 °C and the aqueous phase was moved to a new tube. A 0.4× volume of Ampure XP beads (Fisher Scientific A63881) were added to 100 µL of aqueous phase. They were rotated for 2 min at RT and placed on a magnet until the beads had settled. The supernatant was moved to a new tube and quantified via nanodrop. Small RNAs were isolated from 100 µg of this supernatant using a Zymo Research RNA Clean and Concentrator kit (R1018) according to the manufacturer’s instructions. Dilution of the bead supernatant for the first step of the kit was done with 10 mM NaOAc pH 4.5, not with water. Small RNA was eluted in 30 µL of 10 mM NaOAc pH 4.5 and quantified via nanodrop. It was stored at −80 °C. Two 3′ DNA-RNA hybrid splint adapters were designed with different internal sequences, one to ligate to deacylated tRNAs (“uncharged 3′ adapter”) and the other to acylated tRNAs (“charged 3′ adapter”). A universal 5′ adapter was designed to pair with either of the 3′ adapters. Syntheses of these adapters were ordered from IDT and resuspended in water to a concentration of 2 mM. Adapters were run on a 1.5 mM 6% TBU (Tris, boric acid EDTA) V16 polyacrylamide gel with 10 nmol loaded per lane (10-well comb). Staining was not performed and UV shadowing was used to excise the adapters. Gel slices were cut into small pieces and rotated end over end in crush + soak buffer (300 mM NaOAc pH 5.5, 1 mM EDTA pH 8.0, 0.1% SDS) overnight at 4 °C. Nucleic acids were precipitated with 100% ethanol and resuspended in water to a final concentration of 100 µM. Gel-purified charged 3′ adapter was incubated with a 500-fold molar excess of both imidazole and EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) for 2 h at 25 °C to imidazolate the 5′ end of the adapter. 30 µL of cold 99.5%+ acetone saturated with perchlorate and 1 mL of cold 99.5%+ acetone were added to precipitate the imidazolated adapter. The sample was incubated for 20 min on dry ice and then centrifuged at maximum speed for 10 min at 4 °C. The supernatant was removed and the pellet was washed twice with 1 mL 1:1 acetone: diethyl ether. The pellet was dried in a speed vacuum and resuspended in 10 mM imidazole pH 7.0 to a final concentration of 200 µM. Small RNA (15-50 pmol) was incubated in 100 mM MES pH 5.5, 2.5 mM MgCl2, a fivefold molar excess of both imidazolated 3′ adapter and gel-purified 5′ adapter, and 50 mM HEI pH 6.5 for 30 min at 25 °C, establishing a phosphoramidate covalent linkage between the 3′ splint adapter and aminoacylated tRNAs. Ligated products were purified by crush and soak (0.3 M NaOAc pH 5.5, 1 mM EDTA pH 8.0, 0.1% SDS) at 4 °C, overnight) from a 10% TBU polyacrylamide gel, isolating the regions between 70 and 150 nts. The eluate was precipitated by addition of ethanol and GlycoBlue coprecipitant (Invitrogen) resuspended in a small volume of 10 mM NaOAc pH 4.5 and quantified via absorbance at 260 nm (Nanodrop). Gel-purified tRNAs from chemical ligation were enzymatically ligated to capture deacylated tRNAs with 3′ splint adapters and attach 5′ adapters to all tRNA. tRNA from the first ligation (20 pmol) was incubated in a 20 µL reaction consisting of 10% PEG 8000, 1 µL of RNase inhibitor (Watchmaker Genomics), 9 pmol gel-purified uncharged 3′ splint adapter, 9 pmol gel-purified 5′ adapter, 1× T4 RNA ligase 2 buffer, and 2 µL of T4 RNA ligase 2 (homemade preparation, 0.74 mg/mL). This ligation was incubated at 25 °C for 30 min. Ligation products were purified by addition of a 1.8× volume of tRNA beads (BioDynami), mixing by pipetting, and incubation on ice for 4 min, followed by magnetic separation. The supernatant was discarded. Beads were washed with 180 µL 80% EtOH and air dried. Beads were resuspended in 13 µL of water, and the elution was moved to a new tube and quantified. Splint-adapter-ligated tRNAs are next ligated to RT adapters (RTA) (provided in the RNA004 ONT kit): 12.5 µL of sample was incubated with 1.5 µL RTA, 0.5 µL RNase inhibitor (Watchmaker Genomics), 4 µL T4 DNA ligase buffer, and 1.5 µL T4 DNA ligase (Watchmaker Genomics, 600 U/µL) for 30 min at 25 °C, and cleaned up at RT using the tRNA beads as above, using a 1.35× volume of beads, and elution in 26 µL water. Each sample was quantified with the Quant-iT Qubit dsDNA HS kit and the library size distribution was confirmed by Agilent TapeStation (HS DNA 1000). Finally, ligation products were ligated to ONT’s RNA ligation adapter (RLA) on the same day that sequencing was conducted. 50–400 fmol of sample in 23 µL was incubated with 6 µL RLA (ONT RNA004), 8 µL T4 DNA ligase buffer, and 3 µL T4 DNA ligase (Watchmaker Genomics, 600 U/µL) for 30 min at 25 °C. These final ligations were cleaned up using a 1.8× volume of Ampure XP SPRI beads (Beckman Coulter), and washed with WSB wash buffer (ONT) following the protocol for ONT SQK-RNA004. |
| Analysis protocol | Libraries were basecalled with Dorado v0.7.2 (ONT, https://github.com/nanoporetech/dorado) using the “super high accuracy” (rna004_130bps_sup) v5.0.0 model and –emit-moves parameter. Basecalled bams were converted to fastq format using samtools v. 1.2187 with -T “*” flag to retain move tables, and then aligned to using BWA-MEM version 0.7.16-r118188 with the parameters bwa mem –C –W 13 –k 6 –x ont2d, enabling transfer of move tables to aligned bams. Signal metrics (dwell time and trimmed means of ionic current) at reference-anchored positions were extracted from POD5 files using the Remora API and stored in TSV files for analysis and plotting in R. |
| Organism/cell line | S. cerevisiae |
| Conditions | Loss of specific tRNA modification enzymes |
| Approximate experimental time | 3-4 days |
| Starting RNA amount | 100 µg total RNA |
| tRNA expression | ++ |
| Base modifications | ++ |
| Charging status | ++ |
| tRNA processing and fragmentation | - |
| Citation | https://doi.org/10.1038/s41467-025-62545-9 |
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