Modifications and functional genomics of human transfer RNA

Abstract

Transfer RNA (tRNA) is present at tens of millions of transcripts in a human cell and is the most abundant RNA in moles among all cellular RNAs. tRNA is also the most extensively modified RNA with, on an average, 13 modifications per molecule. The primary function of tRNA as the adaptor of amino acids and the genetic code in protein synthesis is well known. tRNA modifications play multi-faceted roles in decoding and other cellular processes. The abundance, modification, and aminoacylation (charging) levels of tRNAs contribute to mRNA decoding in ways that reflect the cell type and its environment; however, how these factors work together to maximize translation efficiency remains to be understood. tRNAs also interact with many proteins not involved in translation and this may coordinate translation activity and other processes in the cell. This review focuses on the modifications and the functional genomics of human tRNA and discusses future perspectives on the explorations of human tRNA biology.

Fig. 1

tRNA^Ala(AGC) gene sequence diversity. Only genes with tRNAScan score >50 are shown. The four helices are connected by lines. Anticodon nucleotides are marked in bold. The number of genes with identical sequences is in parenthesis. The sequence difference compared to the tRNA with the highest number of gene copies is in red. a The human reference genome has 22 genes, 15 sequences. b C. elegans has 19 genes, 3 sequences. c D. melanogaster has 12 genes, 2 sequences. d S. cerevisiae has 11 genes and 1 sequence

Fig. 2

Human tRNA^Arg(TCT) gene sequences. The four helices are connected by lines. Anticodon nucleotides are marked in bold. The number of genes with identical sequences is in parenthesis. a Five “house-keeping” isodecoder genes. All have introns shown in parenthesis with the number of intronic nucleotides indicated. Sequence differences are in red. b The central nervous system isodecoder gene. It has no introns. Also shown in blue is the nucleotide between the D stem and anticodon stem, and it is a G in this isodecoder but A in all others

Fig. 3

Human cytosolic tRNA^Tyr has the most known modifications. Modified nucleotides are in red; anticodon nucleotides are underlined. Single letter and symbol designations are the nomenclatures used by the RNA modification databases such as Modomics.^– L N²-methyl-G (m²G), D dihydro-U, X 3-(3-amino-3-carboxypropyl)-U (acp³U), R N²,N²-dimethyl-G (m²₂G), 9 galactosyl-queuosine (gal-Q), P pseudoU (Ψ), K N¹-methyl-G (m¹G);], N¹-methylpseudo-U (m¹Ψ), 7 N⁷-methyl-G (m⁷G), ? 5-methyl-C (m⁵C), T 5-methyl-U (m⁵U), “ N¹-methyl-A (m¹A)

Fig. 4

Human nuclear-encoded tRNA modifications investigated by mutation and stop signatures in demethylase-tRNA-seq (DM-tRNA-seq). m¹A N¹-methyl-A, m¹I N¹-methyl-inosine, I inosine, ms²t⁶A 2-methylthio- N⁶-threonylcarbamoyl-A, m¹G N¹-methyl-G, m²₂G N²,N²-dimethyl-G, m³C 3-methyl-C, acp³U 3-(3-amino-3-carboxypropyl)-U

Fig. 5

Watson–Crick face methylations in tRNA^Arg(UCU) isodecoders. Data from ref. . +DM demethylase-treated, m¹A58* only mutation signature was measured for this modification due to the challenge of mapping stops derived from short reads. a An isodecoder from the “house-keeping” group of five intron-containing genes. b The single, central nervous system-specific isodecoder

Fig. 6

Working model for tRNA m¹A58 demethylase (eraser) function. Modified tRNA has a higher affinity for the EF-1A protein, which delivers tRNA to the ribosome. Elevated ALKBH1 activity decreases tRNA availability for translation but may enhance tRNA binding to other cellular proteins due to reduced competition by EF-1A

Fig. 7

Dynamics of protein synthesis needs and tRNA response. During cell cycle progression, most translation activity is for the synthesis of highly abundant house-keeping proteins (~10–20% of all genes), and the tRNA abundance matters most. In stressful times or environmental fluctuations, translation activity switches to the synthesis of response or regulatory proteins (30–50% of all genes), to which tRNA charging significantly contributes. Modification is the “wild card” that tunes the regulation under all conditions

Fig. 8

tRNA-charging level changes upon threonine starvation. Starvation of MDA-MB-231 cells was performed by exchanging the growth medium with the same medium lacking the amino acid threonine at t = 0. Total RNA was isolated at the indicated time points under mild acidic conditions (pH ~ 5) where tRNAs remained aminoacylated. Relative charging levels were measured using tRNA microarrays at the isoacceptor resolution.^, a Nuclear-encoded tRNA^Thr and tRNA^Ser. Thr-mGT corresponds to an array probe complementary to all Thr-AGT and three of the five Thr-CGT tRNA genes. Thr-CGT corresponds to two of the five Thr-CGT tRNA genes. b Mitochondrial-encoded tRNA^Thr and tRNA^Ser. Inset shows the expanded view for early time points. Upon starvation, charging levels for all tRNA^Thr isoacceptors immediately dropped but to different degrees. Differential charging remained for several hours. Mitochondrial tRNA^Thr recovered more slowly than the cytosolic tRNA^Thr. Charging levels did not drop for tRNA^Ser

Fig. 9

DM-tRNA-seq can measure tRNA abundance, modification, and charging in a single-sequencing library. Periodate only oxidizes uncharged tRNA. β-Elimination removes the oxidized 3′ nucleotides and deacylates charged tRNAs at the same time. The sample is then split into two, and one part treated with demethylases to remove m¹A, m³C, and m¹G (orange circles). Both samples are subjected to cDNA synthesis by a processive RT with both dT/dG-ending template-switching primers in case of TGIRT. cDNAs are circularized, followed by PCR to build sequencing libraries