| Summary | tRNA sequencing using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase |
| Key findings | DM-tRNA-seq allows for quantitative analysis of tRNA abundance and modifications (including m1G37, m1A58, m3C32, m1G9) in a high throughput manner |
| Sequencing strategy | RNA-Seq |
| Raw data | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66550 |
| Experimental protocol | Total RNA was isolated by using a mirVana miRNA Isolation Kit (Life Technologies). Purified total RNA was premixed with the T7 RNA polymerase transcripts of three tRNA standards13 (0.01 pmol each standard per μg of total RNA) and deacylated by incubating in 0.1 M Tris-HCl, pH 9 at 37°C for 45 min. Although not necessary for studies of mature tRNAs, which all end with 3’CCA, deacylated RNAs with or without demethylation treatment could be treated with T4 polynucleotide kinase (Epicentre) at 37C° for 30 min to further warrant a free 3’ hydroxyl group for template-switching. When necessary, total tRNA was subsequently isolated using a denaturing 10% polyacrylamide gel followed by passive gel elution and ethanol precipitation. Demethylation activity assay was performed for either gel purified total tRNA or total cellular RNA. For total tRNA, 100 μl of reaction mixture containing 1 μg of tRNA (~40 pmol) was treated with 2× molar ratio of wtAlkB (80 pmol) and 4× molar ratio of D135S mutant (160 pmol). For total cellular RNA, 5 μg of total RNA (estimated to contain ~40 pmol tRNA) was treated with 4× molar ratio of wtAlkB (160 pmol) and 4× molar ratio of D135S (200 pmol). More demethylases were used for total RNA to alleviate potential interference by rRNA and mRNA in the reaction. The reaction buffer contained 300 mM KCl, 2 mM MgCl2, 50 μM of (NH4)2Fe(SO4)2·6H2O, 300 μM 2-ketoglutarate (2-KG), 2 mM L-ascorbic acid, 50 μg/mL BSA, 50 mM MES buffer (pH 5.0). In both cases, the reaction was incubated for 2 hr at room temperature, and quenched by the addition of 5 mM EDTA. After phenol–chloroform extraction, tRNA was recovered by ethanol precipitation. Template-switching reactions were performed using an initial template-primer substrate consisting of a 41-nt RNA oligonucleotide (5’-AGA UCG GAA GAG CAC ACG UCU AGU UCU ACA GUC CGA CGA UC/3SpC3/-3’) that contains Illumina Read1 and Read2 primer-binding sites and a 3’ blocking group (three carbon spacer; Integrated DNA technologies, inc.) annealed to a complementary 32P-labeled DNA primer with a single-nucleotide 3’ overhang, T, which facilitates the template switch to full-length tRNAs that mostly contain a 3’ CCA end. For TGIRT template-switching reactions, typically 100 ng of demethylated tRNAs or 1 μg of demethylated total RNA were mixed with the initial template-primer substrate (100 nM) and 500 nM TGIRT (GsI-IIC MalE rigid fusion RT) in reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, and 5 mM DTT. The reactions were pre-incubated at room temperature for 30 min, initiated by adding 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP and dTTP) to a final concentration of 1 mM and incubating at 60°C for 30 min. The reactions were terminated by adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95°C for 3 min, and neutralizing with 5 M HCl. The cDNAs resulting from template-switching were analyzed in a denaturing 6% polyacrylamide gel, electroeluted using a D-tube Dialyzer Maxi with MWCO of 6–8 kDa (EMD Millipore), and ethanol precipitated with 0.3 M sodium acetate, pH 5.2, in the presence of 25 μg of linear acrylamide (Life Technologies) carrier. The purified cDNAs were then circularized with CircLigase II (Epicentre) using the manufacturer's protocol with an extended incubation time of 5 hr at 60°C, extracted with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated and amplified with Phusion-HF (Thermo Scientific) using Illumina multiplex (5’- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3’) and barcode (5’- CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T -3’) primers for 12 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The PCR products were sequenced on an Illumina HiSeq system. |
| Analysis protocol | Sequencing reads were aligned using Bowtie to a modified hg19 genomic tRNA database. A single mismatch was allowed in order to identify potential modification misincorporations at a modification site. Briefly, a tRNA library was adapted from the tRNAScan-SE library by appending CCA to tRNAs from the genomic tRNA database (http://gtrnadb.ucsc.edu/Hsapi19/). Isodecoders with identical scores were consolidated for ease of identity assignment, decreasing the number of reference genes and pseudogenes from 625 to 462. Prior to mapping, reads were processed using Trimmomatic v0.32. Sequences greater than 15 bp were then aligned to the aforementioned culled tRNA library using Bowtie2 with sensitive options. Reads mapping to multiple isodecoders due to length of fragment were discarded. Modification fractions were determined by analyzing at a putative position ‘n’ the number of correct reads, the number of misincorporations, as well as the number of reads stopped at the n+1 position. |
| Organism/cell line | Homo sapiens (HEK293T) |
| Conditions | demethylase treated and untreated |
| Approximate experimental time | 1-2 days |
| Starting RNA amount | 5 µg total RNA |
| tRNA expression | ++ |
| Base modifications | + |
| Charging status | - |
| tRNA processing and fragmentation | - |
| Citation | https://doi.org/10.1038/nmeth.3478 |
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