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1038/s41568-023-00611-4

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Transfer RNAs as dynamic

and critical regulators of
cancer progression
Alexandra M. Pinzaru & Sohail F. Tavazoie
Abstract Sections

Transfer RNAs (tRNAs) have been historically viewed as non-dynamic Introduction

adaptors that decode the genetic code into proteins. Recent work Variation in tRNA pools and its
has uncovered dynamic regulatory roles for these fascinating relevance to cancer

molecules. Advances in tRNA detection methods have revealed that Genetic and transcriptional
tRNA deregulation in cancer
specific tRNAs can become modulated upon DNA copy number
and chromatin alterations and can also be perturbed by oncogenic Altered tRNA stability
in cancer
signalling and transcriptional regulators in cancer cells or the
Microenvironmental
tumour microenvironment. Such alterations in the levels of specific influences on tRNAs in cancer
tRNAs have been shown to causally impact cancer progression,
Roles of aminoacyl-tRNA
including metastasis. Moreover, sequencing methods have identified synthetases in cancer
tRNA-derived small RNAs that influence various aspects of cancer
Functions of tRNA fragments
progression, such as cell proliferation and invasion, and could serve in cancer
as diagnostic and prognostic biomarkers or putative therapeutic Conclusions and future
targets in various cancers. Finally, there is accumulating evidence, perspectives

including from genetic models, that specific tRNA synthetases — the

enzymes responsible for charging tRNAs with amino acids — can either
promote or suppress tumour formation. In this Review, we provide an
overview of how deregulation of tRNAs influences cancer formation
and progression.

Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA. e-mail: apinzaru@
rockefeller.edu; sohail.tavazoie@rockefeller.edu

Nature Reviews Cancer

Review article
Introduction
Transfer RNAs (tRNAs) are small non-coding RNAs that are essential
to protein synthesis, acting as the adaptor molecules that translate
the codon information in messenger RNAs (mRNAs) into the amino
acid sequences of proteins. First postulated by Francis Crick in his
‘adaptor hypothesis’, tRNAs were discovered in the late 1950s in bio-
chemical studies1–3. Decades of research elucidated the central role that
tRNAs have in protein synthesis and provided a foundation for RNA
research more broadly. tRNA studies inspired the development of many
techniques that are now commonly used to study other types of RNA
and RNA–protein interactions, such as methods to structurally map
RNAs and RNA–protein photocrosslinking4. Human cells have two dis-
tinct pools of tRNAs: a cytosolic pool encoded by nuclear tRNA genes,
and a mitochondrial pool encoded by 22 mitochondrial tRNA (mt-tRNA)
genes. True to their postulated evolutionary origins, mt-tRNAs share
features with bacterial tRNAs that deviate from the cytosolic tRNAs,
with peculiarities in their genetic code and secondary structures. Here
we focus on cytosolic tRNA (hereafter referred to as tRNA) and we refer
the reader to recent reviews for detailed discussion on mt-tRNA5–7.
The main function of tRNAs is to accept activated amino acids
from aminoacyl-tRNA synthetases (aaRSs) and subsequently transfer
them to a growing peptide chain at the ribosome upon specific codon
recognition during protein translation. This function is achieved due
to the unique structural features of tRNAs. In eukaryotes, tRNAs are
short nucleic acid sequences, approximately 73–100 nucleotides, that
fold into a ‘cloverleaf’ secondary structure8 and an L-shaped tertiary
structure9,10 (Fig. 1a). One arm of the ‘L’ contains the aminoacylation site,
whereas the second arm includes the three-nucleotide anticodon neces-
sary for codon recognition on mRNAs (Fig. 1a). To ensure an adequate
supply of tRNAs that match the proteomic needs of a cell, tRNAs are tran-
scribed by the specialized RNA polymerase III (Pol III)11 (Fig. 1b). Canoni-
cally, tRNA genes contain internal promoter sequences (Fig. 1b), except
for the selenocysteine tRNA gene, which uses extragenic promoter
elements, including a TATA box upstream of the transcription start
site11. Following transcription, the produced precursor tRNA (pre-tRNA)
undergoes extensive processing, including removal of 5′ leader and 3′
trailer sequences, 3′-CCA addition and common modifications, as well
as quality control to generate a functional mature tRNA12 (Fig. 1c).
For many decades, owing to their essential function in translation,
tRNAs were considered to be static molecular effectors in cells. However,
data from mass spectrometry, hybridization-based techniques such as
microarrays, next-generation sequencing approaches and Pol III occu-
pancy analysis have uncovered a dynamic pattern of tRNA expression
that is dependent on tissue type, developmental stage and prolifera-
tive state, and have revealed that tRNAs can have regulatory roles and
respond to environmental cues to shape gene expression13–21. The grow-
ing appreciation of tRNAs as important post-transcriptional regulators
of gene expression, aided by an expanding toolkit to study and manipu-
late tRNA, has sparked great interest in the past 15 years to uncover
the previously unexplored roles of tRNA in cancer. In this Review, we
describe loss-of-function, gain-of-function and clinical association
studies that provide evidence for canonical and non-canonical roles of
tRNAs in cancer. We also detail recent findings suggesting that aaRSs
can act as tumour suppressors or promoters of cancer progression.
Variation in tRNA pools and its relevance to cancer
A distinctive and fascinating feature of tRNAs is their extensive incor-
poration of modified nucleosides, which are crucial for tRNA stability
and function. Of the approximately 150 ribonucleoside modifications
Nature Reviews Cancer
that have been identified so far, approximately 80% occur in tRNAs22.
These modifications, the most common of which are base methy­
lations, modulate the affinity of the anticodon–codon interaction, help
to maintain the fidelity of the open reading frame, ensure correct tRNA
recognition and discrimination by aaRSs, and impact tRNA folding
and stability (reviewed in refs. 5,23–25). In eukaryotes, it has been esti-
mated that each tRNA molecule contains on average 13 modifications23.
The types, patterns and extent of modifications differ between tRNA
species; even within the same tRNA isotype, the levels of modifica-
tion of a specific base can vary substantially depending on cellular
conditions5,23,26. This generates complexity within tRNA pools, as cells
with the same tRNA transcriptional expression patterns can vary in
terms of the composition of their functional tRNA pools depending
on their modification status and cellular conditions. An important
source of functional variation in tRNAs comes from modifications
that modulate the decoding ability of tRNAs. For example, inosine,
a deaminated adenosine, can pair with adenosine (A), cytidine (C)
and uridine (U), and its presence at position 34 in the anticodon of
the yeast alanine tRNA8 prompted Francis Crick to develop the ‘wobble
hypothesis’27. Wobble pairing expands the decoding ability of tRNAs,
allowing all 61 codons corresponding to the standard 20 amino acids
to be decoded even though certain codons are missing cognate tRNA
genes, as humans have only 47 tRNA families with distinct anticodons
according to current in silico predictions28 (Table 1).
The human genome encodes approximately 619 predicted tRNA
genes, out of which 429 are of high confidence, meaning they pass all
the filtering criteria of the current prediction algorithm that distin-
guishes bona fide tRNA genes that function in protein translation from
tRNA-derived repetitive elements in the human genome28,29 (Table 1).
tRNAs that carry the same amino acid but have different anticodons
are called isoacceptors. Furthermore, an isoacceptor family consists
of a variable number of isodecoders, which are tRNAs that share the
same anticodon, but have sequence differences in the body of the tRNA.
These features contribute to the large diversity of tRNA sequences
and the complexity of the tRNAome, the regulation of which remains
an active area of investigation. The number of isodecoders a given
isoacceptor family can have varies greatly. For example, the tRNACysGCA
isoacceptor family comprises 23 different isodecoders, encoded by
29 genes, whereas the tRNAHisGTG isoacceptor has only one isodecoder
that is encoded by 9 duplicated genes28 (Table 1). This tRNA diversity
is thought to have evolved to match the dynamic proteomic needs of a
complex organism such as a human, and deregulation of this diversity
is implicated in disease. For example, tRNA levels have been found to
be altered in cancer cells compared with normal cells, probably as
a means to optimize protein production to sustain the growth and
survival of cancer cells18,30. Moreover, as detailed below, there is evi-
dence of specific tRNAs regulating cell proliferation31 and metastatic
progression21,32, functionally implicating tRNAs in various steps of
cancer initiation and progression (Fig. 2).
Functional consequences of altered tRNA pools in cancer
In the late 1960s, chromatography-based techniques began hint-
ing at differences in the levels of tRNAs between tumour cells and
untransformed cells33–35. However, it was not until 2009 that a reliable
genome-wide assessment of tRNA expression in cancer was reported30.
Using microarrays to assess the levels of 40 nuclear-encoded isoac-
ceptors in normal and cancerous breast epithelial cells as well as
patient-derived tumours, the authors found a consistent trend of over-
expression of tRNAs in breast cancer cell lines and tissues relative to
Review article

normal controls. In addition, a selective upregulation of certain tRNA genes30. A later study from a different group used custom-made micro-
isoacceptors was detected, including tRNAArgCCU, tRNAThrCGU, tRNALeuUAA, arrays to profile in parallel the levels of 206 tRNA genes and approxi-
tRNATyrGUA, tRNASerGCU and tRNAArgUCU, which the authors speculated mately 7,000 mRNAs from an extensive set of primary tumours and
may be necessary for the efficient translation of tumorigenesis-related cancer cell lines, including bladder, colon and prostate cancers, as

a 3′
A
C
C
5′ N73

Acceptor
stem TψC-
TψC- loop
D-loop stem
D-stem

Variable
Anticodon region
stem

Anticodon
loop

Anticodon 34 35 36

b c RNase P RNase Z

3′
5′ Trailer
Pol III TFIIIC Leader All tRNAs
• 5′ Leader and 3′
5′ TBP BRF1 trailer removal
TSS
3′ BDP1 • Common
A box B box TTTTT modifications
tRNA • 3′-CCA addition

Specific tRNAs
• Intron splicing
Intron • Specific
modifications
• Aminoacylation

Fig. 1 | Transfer RNA structure and biogenesis. a, Schematic on the left
shows the classical transfer RNA (tRNA) cloverleaf secondary structure with the
acceptor stem, the variable region and the three arms (each consisting of a stem
and a loop region), including the anticodon arm, the dihydrouridine-containing
(D)-arm, and the thymidine, pseudouridine and cytidine-containing (TψC)-
arm. The anticodon loop includes in its centre, at positions 34–36 (5′ → 3′), the
anticodon necessary for codon recognition on mRNAs. The 3′ end of the acceptor
stem ends in an invariant CCA triplet that contains the aminoacylation site on
the final adenosine167 with an upstream discriminator base (N73), which is used
as a recognition element by aminoacyl-tRNA synthetases168. The acceptor stem,
anticodon arm and the TψC-arm have conserved sizes, whereas the D-arm and the
variable loop sizes can differ, accounting for the variation in tRNA length167,169 (not
shown). On the right is shown the tertiary structure of a tRNA based on the crystal
structure of yeast phenylalanine tRNA170 (Protein Data Bank ID: 1EHZ) (the 3D
rendition was created using the UCSF ChimeraX 1.4 software). Base stacking and
interactions between conserved or semi-conserved residues, mainly between the
D-loops and TψC-loops, lead to the 3D L-shape of the tRNA. The TψC-stem stacks
onto the acceptor stem to form one arm of the ‘L’, whereas the anticodon stem–
loop stacks onto the D-stem to form the second arm of the ‘L’. The colour scheme
matches the one used in the secondary structure. b, tRNA genes contain internal
promoter sequences called the A box and B box171,172 that are recognized by the
multisubunit general transcription factor IIIC (TFIIIC), which then recruits
the TFIIIB transcription factor, a trimeric complex composed of TATA-box binding
protein (TBP), transcription factor TFIIIB component B″ homologue (BDP1) and
transcription factor IIIB 90 kDa subunit (BRF1). DNA-bound TFIIIB and TFIIIC
recruit the specialized RNA polymerase III (Pol III)11 to form the pre-initiation
complex (PIC) at the tRNA genes. Structural rearrangements of the PIC allow
transcription initiation and promoter escape, followed by transcription
elongation until a termination signal, a short stretch of at least five thymidine
nucleotides in human cells on the non-template strand, is encountered. Upon
transcription termination, Pol III is recycled to rapidly reinitiate transcription
of the same gene, resulting in high yields of tRNA transcripts11. c, Processing of
the precursor tRNA (pre-tRNA) involves cleavage of the extra 5′ leader and 3′
trailer sequences by the ribonucleoprotein complex ribonuclease P (RNase P)
and RNase Z12 (for which two forms in human cells, ELAC1 and ELAC2, have been
identified173), respectively. In humans and most eukaryotes, tRNA nucleotidyl
transferase 1 (TRNT1) adds the invariant 3′-CCA triplet after the discriminator
base (not shown). A subset of tRNAs contain intronic regions, which are excised
by splicing enzyme complexes, before the tRNAs undergo final modifications
that allow them to assume a functional tertiary structure12. Maturation of tRNAs
includes incorporation of common modifications, such as pseudouridine and
dihydrouridine, whereas certain tRNAs incorporate specific modifications
such as anticodon modifications inosine and queuosine5,12. Finally, each mature
tRNA will undergo aminoacylation, the process in which the tRNA is covalently
bound by a dedicated aminoacyl-tRNA synthetase to a specific amino acid
corresponding to its anticodon108. TSS, transcription start site.

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Table 1 | High-confidence tRNA genes and corresponding isodecoders (hg38)a

Isoacceptor Anticodon Number of tRNA Number of distinct Isoacceptor Anticodon Number of tRNA Number of distinct
genes isodecoders genes isodecoders

Ala AGC 26 17 Tyr GTA 13 9

CGC 4 4 Val AAC 9 5
TGC 8 7 CAC 13 7
Arg ACG 7 2 TAC 5 4
CCG 4 2 iMet, initiator Met; tRNA, transfer RNA. aData curated from GtRNAdb28.

TCG 6 6
well as glioblastomas and diffuse large B cell lymphomas (DLBCLs)18.
CCT 5 5
The authors compared tRNA pools in cancer samples relative to nor-
TCT 6 5 mal tissue controls and found that patients within the same cancer
Asn GTT 25 16 type tended to have similar tRNA pools, but that similarity decreased
when comparing different cancer types to each other. Moreover, by
Asp GTC 13 3
experimentally altering cell proliferation and differentiation in vari-
Cys GCA 29 23
ous cellular systems, the authors identified ‘differentiation tRNAs’ and
Gln CTG 13 7 ‘proliferation tRNAs’, the tRNA availability of which was correlated with
TTG 6 4 the distinct codon usage of proliferation genes versus differentiation
genes. In addition, the authors used their DLBCL data to detail specific
Glu CTC 8 2
tRNA changes and reported that, relative to normal B cells, DLBCL
TTC 8 5 samples exhibited consistent changes in their tRNA pools, with several
Gly GCC 14 5 tRNAs showing over tenfold enrichment, such as tRNALysCUU, whereas
CCC 5 3
other tRNAs exhibited more than tenfold depletion, such as tRNATyrGUA.
Finally, across different cancer types, the authors also revealed correla-
TCC 9 4
tions between the levels of upregulated tRNAs in cancer samples with
His GTG 9 1 the codon usage of transcripts induced in cancer18.
Ile AAT 15 10 The early explorations of the tRNA profiles in cancer cells relative
to normal cells have provided important clues to how tRNA mole­
GAT 3 1
cules might affect cancer progression. However, loss-of-function
TAT 5 3 and gain-of-function analyses are necessary to establish causative
iMet/Met CAT 9/11 2/7 roles for tRNA changes during tumorigenesis (Box 1). One of the first
Leu AAG 9 4 tRNAs to be experimentally modulated was the initiator methionine
(tRNAiMet), which has an essential role during translation initiation —
CAG 9 2
the rate-limiting step during protein synthesis36 — and is upregulated
TAG 3 3 in breast cancer samples30. It has been shown that the overexpression
CAA 6 5 of mature tRNAiMet in untransformed human breast epithelial cells
increased metabolic activity and proliferation in those cells31. The
TAA 4 4
mechanism underlying this observation was not investigated, but
Lys CTT 15 10 the authors reported that overexpressing tRNAiMet increased the levels
TTT 12 8 of other tRNAs, emphasizing the complexity of tRNA regulation31. In a
Phe GAA 10 5 different study, overexpression of tRNAiMet in a highly metastatic human
melanoma cell line did not affect primary tumour growth but increased
Pro AGG 9 2
migration and invasion of cancer cells37. These studies have revealed
CGG 4 2 that overexpression of tRNAiMet promotes tumorigenic phenotypes
TGG 7 3 consistent with enhancement of global translation eliciting malignant
SelCys TCA 1 1
phenotypes38,39. Furthermore, these results also suggest that the spe-
cific effects of tRNA overexpression on cancer progression may be con-
Ser AGA 9 4
text dependent, causing different phenotypes depending on cell type.
CGA 4 4 In addition to cancer cells, increased levels of tRNAiMet have also been
TGA 4 4 reported in cancer-associated fibroblasts, and the potential effects
of this dysregulation in the tumour stroma have been investigated
GCT 8 6
by introducing two copies of the gene encoding tRNAiMet upstream of
Thr AGT 9 6 exon 1 of the Hprt gene in a mouse model (2 + tRNAiMet mice). The authors
CGT 5 5 found that syngeneic tumours injected into 2 + tRNAiMet mice grew
TGT 6 6
faster and exhibited greater vascularization than tumours injected
into littermate controls. These tumour phenotypes were linked to
Trp CCA 7 5
preferential altering of the secretome, as mouse embryonic fibroblasts

Nature Reviews Cancer

Review article
derived from the 2 + tRNAiMet mice exhibited increased production and
secretion of collagen proteins40. These studies have suggested that
tRNAiMet can promote specific tumorigenic phenotypes in a cell-type-
dependent manner (Fig. 2) and raise the question of how tRNAiMet levels
are regulated in cancer. As detailed below, such regulation may be both
transcriptional41 and post-transcriptional42.
To probe the question of whether specific tRNA alterations could
change distinct downstream translational programs during cancer
progression, a hybridization-based, high-throughput tRNA profiling
method was developed to investigate tRNA dynamics during cancer
progression21. Unbiased quantification of isoacceptor levels revealed
that tRNAGluUUC and tRNAArgCCG levels were consistently upregulated
in highly metastatic breast cancer cells compared with their isogenic
parental lines21. Depletion and overexpression studies found that these
two tRNAs promoted the invasiveness and enhanced the metastatic
capacity of breast cancer cells. Moreover, tRNAGluUUC and tRNAArgCCG
levels were observed to be upregulated in metastatic primary human
Leu
Normal cell
CAG
1 Transformation
Arg
UCU
Cancerous cell
iMet (poorly metastatic)
CAT
2 Tumour Metastasis 3
growth
↑ Translation
? Specific
↑ Proliferation proteins
Fig. 2 | Roles of mature transfer RNAs in oncogenic transformation and
cancer progression. Model showing how mature transfer RNA (tRNA) levels
can influence tumour initiation and progression. Shown are examples of
tRNAs reported to be involved at each stage during cancer progression.
Oncogenic transformation (1) can be suppressed in oncogene-expressing breast
epithelial cells by tRNALeuCAG (ref. 103), or promoted in simian virus 40 (SV40)
immortalized mouse embryonic fibroblasts or in NrasG12D/+ haematopoietic stem
or progenitor cells by ectopic expression of tRNAArgUCU (ref. 81). Tumour growth
(2) has been shown to be influenced by various tRNAs18,30,31,40,81–85. Increased
tRNAiMet levels can promote mRNA translation174 with cell-type-specific effects,
Nature Reviews Cancer
breast tumours compared with non-metastatic primary tumours,
revealing that their expression correlated with human breast cancer
metastatic potential. Integrating data from ribosome profiling, prot-
eomics and whole-genome measurements of mRNA stability revealed
that tRNAGluUUC and tRNAArgCCG overexpression specifically promoted
the translation of mRNAs enriched in codons cognate to the overex-
pressed tRNAs. Two such genes, exosome component 2 (EXOSC2) and
GRIP1-associated protein 1 (GRIPAP1), were identified as downstream
mediators of the pro-metastatic effects of tRNAGluUUC21. Together, this
study demonstrated that upregulation of specific tRNAs can not only
regulate specific pathways but also causally drive invasive and meta-
static phenotypes through enhanced translation of a specific down-
stream gene network comprising genes enriched in the codons cognate
to the upregulated tRNAs (Fig. 2).
Despite accumulating evidence that tRNAs can regulate cancer
progression, systematic studies that detail changes in tRNA levels dur-
ing different stages of cancer evolution across different cancer types
Glu Arg Ile Ile
UUC CCG GAU UAU
Ribosome
CGG rich
mRNA AUC rich
GAR rich
AUA rich
Metastatic
progression
Metastatic
progression
including increased proliferation31 by unknown mechanisms (indicated by ‘?’).
In mouse cancer-associated fibroblasts, tRNAiMet upregulation leads to
increased production of specific proteins, which increases tumour growth40.
During metastatic progression (3), altered levels of specific tRNAs promote the
translation of the mRNAs enriched in the codons cognate to the upregulated
tRNAs21,32. These can include enhanced translation of GAR-rich (GAA and
GAG codons) or CGG-rich mRNAs in response to upregulation of tRNAGluUUC
and tRNAArgCCG21 or preferential translation of AUA-rich over AUC-rich
mRNAs in response to changes in the ratio of tRNAIleGAU to tRNAIleUAU (ref. 32).
mRNA, messenger RNA.
Review article
are still lacking. One limitation of such studies still lies in achieving
accurate tRNA quantification due to the intricate modification pat-
terns on tRNAs that block reverse transcription, their rigid secondary
structures as well as high sequence similarity. Even though absolute
Box 1
Challenges in measuring and experimentally modulating tRNA
levels
Historically, one of the major hurdles in studying transfer RNAs
(tRNAs) in cancer has been obtaining high-resolution, accurate
quantification of tRNA levels23. Next-generation sequencing offers
accessible, relatively inexpensive platforms for quantifying tRNAs,
but library preparation for next-generation sequencing relies on
reverse transcription of the tRNA molecules. The stable folding
and modification of tRNAs cause the reverse transcriptase to stop
before reaching the end of the molecules, resulting in many short,
incomplete reads, which preclude accurate quantification of mature
tRNA levels. This problem is compounded by the high sequence
similarity of tRNA genes, which can differ by only a few nucleotides
for different isoacceptors28, meaning the short reads can map to
multiple tRNA genes. For these reasons, we caution against the
inference of mature tRNA levels from small-RNA sequencing data
not geared towards full-length tRNA expression identification, such
as small-RNA sequencing datasets from The Cancer Genome Atlas,
which are specifically aimed at identifying microRNAs and have read
lengths of only approximately 30 nucleotides175.
Different strategies for overcoming the reverse transcription
blocks have emerged, including using higher processivity reverse
transcriptases that can read through some of the modifications
and/or by enzymatically removing certain modifications altogether,
where possible23,176,177. Interestingly, next-generation sequencing-
based approaches have also been developed to detect and quantify
a subset of tRNA modifications23,44. Some tRNA profiling methods use
hybridization approaches to avoid the reverse transcription step
altogether15,21, but they lack the sensitivity to distinguish between
tRNA genes that differ by less than eight nucleotides15. An approach
that holds high promise to retrieve both the full tRNA sequences
and their modification status (but not necessarily the identity of the
modification) is direct sequencing using nanopore sequencing
technology46,178,179. This is a developing technology that has very
recently been shown in yeast to have potential for tRNA quantifica­
tion46 and — when optimized for human tRNA — might streamline
detection of tRNA levels in cancer samples.
To show causal roles for tRNAs in cancer, it is critical to generate
the appropriate loss-of-function or gain-of-function models of
disease. However, tRNAs present unique challenges in achieving
robust modulation of their levels. As opposed to most coding genes,
which have just two alleles, all tRNAs except tRNASec are encoded
by multigene families (Table 1), challenging attempts to create tRNA
isotype knockouts, especially when generating mouse models, as one
may need to edit several genomic loci at once to achieve sufficient
depletion of the tRNA of interest. This was recently exemplified in
a study using CRISPR–Cas9 to generate single and combinatorial
Nature Reviews Cancer
quantification of tRNAs is still not possible, recent strides have been
made in circumventing some of the hurdles imposed by tRNA modi-
fications and provide improved relative tRNA quantification at the
isodecoder level16,43–46 (Box 1 and reviewed in ref. 23).
gene knockouts for all seven murine isodecoder genes encoding
tRNAPheGAA (ref. 180). In line with the expected redundancy of related
isodecoders, the single deletions of six out of the seven isodecoders
did not cause overt phenotypes. Only the single deletion of
tRNA-Phe-GAA-1-1 caused subtle defects in growth and neurological
development180. Knockout of tRNA-Phe-GAA-1-1 worsened the
embryonic lethality phenotype observed upon simultaneous
deletion of multiple tRNA-Phe-GAA isodecoders, suggesting that
not all related tRNA isodecoder genes are functionally redundant.
Interestingly, the reduction of mature tRNAPheGAA in the single
deletion mutants was tissue dependent, with tissues such as the
kidney showing no depletion, whereas others, such as the liver, had
more than 70% reduction in mature tRNAPheGAA levels180. This data,
together with previous reports that different tissues have different
tRNA expression patterns15,16, highlight the need to systematically
investigate the contribution of each tRNA isodecoder gene to the
pool of functional tRNAs for a tissue of interest. Conversely, following
identification of decreased mature tRNA levels in cancer cells, it is
necessary to determine the exact tRNA genes that are downregulated
to properly model them in loss-of-function studies.
Gain-of-function experiments are also complicated, as it can be
challenging to achieve detectable levels of tRNA overexpression31,149.
For example, introducing 10 or 20 copies of tRNA-Gly-GCC in a
Drosophila model with glycyl-tRNA synthetase 1 (GARS1) mutations
only increased mature tRNAGlyGCC levels by approximately 13% and
approximately 25%, respectively, whereas 12 copies of tRNA-Gly-TTC
achieved up to 75% overexpression of tRNAGlyUUC (ref. 149). In mouse
models, two copies of a transgene containing a genomic locus with
two tRNA-Gly-GCC genes was not enough to increase mature tRNA
levels, but expressing approximately 27 copies of that transgene
achieved 90–150% increase in mature tRNAGlyGCC levels, depending
on tissue type149. These results suggest that the exact number of gene
copies necessary to sufficiently increase mature tRNA levels vary
for each tRNA isoacceptor and probably depend on the studied cell
type. In support of that idea, transgenic mice carrying two additional
copies of the gene encoding tRNAiMet had approximately 1.3–1.5-fold
higher levels of tRNAiMet in various tissues40. To note, the integration
of the two copies of the gene encoding tRNAiMet in the Hprt locus in
an open chromatin region overlapping with the Hprt promoter region
upstream of exon 1 might have favoured the transcription of the tRNA
genes40. The use of an upstream, strong RNA polymerase III promoter
such as the U6 promoter has been successfully implemented to
drive higher tRNA gene expression than just by overexpressing the
endogenous tRNA genes181 and may be combined with multiple tRNA
copies to optimize overexpression systems150.
Review article

1 Changes in cancer 2 Effects on tRNA 3 Effects on translation

Mutations: ACTAGAAAT > ACTAGCAAT Altered decoding Amino acid misincorporation

Copy number changes: Polypeptide
Ala Ser Ser Ala
Genetic changes

chain
Ref tRNA-1
Amplifications Ser
tRNA-1 tRNA-1 mRNA
Ala
AGC AGA AGC Ribosome
codon
Ref tRNA-2
Deletions Altered tRNA pools Codon-biased translation
Deletion

DNA Me
MYC
methylation
Transcriptional changes

Pol III TFIIIC

5′ SOX4 TBP BRF1
3′ BDP1 A box B box TTTTT

tRNA

p53 pRb

tRNA
Modification changes degradation
Post-transcriptional
deregulation

Amino acid charging levels

tRNA mischarging Amino acid misincorporation

Trp Phe
Trp
Phe
Amino acid deprivation WARS1
Environmental

CCA CCA Trp codon

stressors

Hypoxia O2
tRNA Non-canonical functions
O2 O2 fragmentation (e.g., regulation of mRNA stability)

Fig. 3 | Regulation of transfer RNA expression in cancer. Diagram summarizing
the processes (1) that can lead to transfer RNA (tRNA) deregulation in cancer
(2), and affect tRNA decoding capabilities, abundance, stability or charging
by aminoacyl-tRNA synthetases resulting in altered messenger RNA (mRNA)
translation (3). BDP1, transcription factor TFIIIB component B″ homologue;
BRF1, transcription factor IIIB 90 kDa subunit; Me, methylation; Pol III, RNA
polymerase III; Ref, reference genome; SOX4, SRY-box transcription factor 4;
TBP, TATA-box binding protein; TFIIIC, general transcription factor IIIC;
TSS, transcription start site; WARS1, tryptophanyl-tRNA synthetase 1.

Genetic and transcriptional tRNA deregulation
in cancer
Copy number variation and mutations in tRNA genes
The observation that tRNAs have prominent roles in tumour pro-
gression raises the question of how tRNA levels are altered in cancer
(Fig. 3). Somatic copy number variation has been shown to be one
mechanism responsible for tRNA upregulation during cancer evolu-
tion to a highly metastatic state21 (Fig. 3). As germline copy number
changes have been reported for certain tRNA genes in humans47,48,
it is tempting to speculate that germline copy number variations of
tRNA genes may influence cancer progression, although their func-
tional implications remain to be investigated. Apart from copy num-
ber variations, tRNA genes exhibit large sequence variation in both
their tRNA bodies49,50 and their immediate flanking regions (approxi-
mately 20 bp upstream and approximately 10 bp downstream from
the mature tRNA)51. Polymorphisms in the body of the tRNA could
impact the efficiency and fidelity of protein translation, as they can
affect the folding, stability and decoding affinity of the tRNA. Indeed,
there are instances in which single tRNA mutations have been found
to contribute to disease phenotypes. For example, a hypomorphic
single-nucleotide polymorphism in one tRNA-Arg-TCT gene caused
ribosome stalling and neurodegeneration in mice when combined
with loss of the GTP-binding protein 2 (GTPBP2) ribosome recycling
factor52. In humans, a loss-of-function homozygous single-nucleotide
polymorphism in the tRNA-SeC-TCA gene preferentially impaired the
production of stress-related selenoproteins, presenting with various
clinical symptoms in a patient53. In proof-of-concept experiments
relevant to cancer, exogenous expression of a tRNASer with a single
point mutation that incorporates serine at specific alanine codons
(tRNASer(Ala)) has been shown to increase the tumorigenic potential of
NIH-3T3 mouse embryonic fibroblasts54. Compared with control cells,
the NIH-3T3 cells expressing tRNASer(Ala) had significantly accelerated
tumour growth in xenografts assays in nude mice. To note, the authors
observed spontaneous transformation of their control NIH-3T3 cells,

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Review article

Glossary genes were occupied across all tested cell lines, these experiments also
identified cell-type-specific patterns of Pol III occupancy in human
cells19,20. Evidence in support of transcriptional control of specific tRNA
Argonaute 2 (AGO2) Ribosome profiling genes during cancer progression was observed for tRNAIleUAU, as highly
cleavage assay A technique that provides a snapshot metastatic breast cancer cells exhibited increased Pol III occupancy
An in vitro assay that uses recombinant of all ribosome-bound messenger RNAs at tRNA-Ile-TAT genes relative to poorly metastatic cells, which resulted
AGO2 to show specific cleavage of in cells with codon-level resolution. in increased tRNAIleUAU expression32. Interestingly, the upregulation
target oligonucleotides. of tRNAIleUAU was accompanied by repression of the related isoacceptor
RNA polymerase III tRNAIleGAU during breast cancer progression32. Mimicking the divergent
Epistatic interactions (Pol III). An RNA polymerase complex modulation of the two related tRNAIle isoacceptors experimentally
Interactions between two or more that translates short, non-coding RNAs, promoted metastatic colonization in breast cancer xenograft models.
genes that influence a particular including transfer RNAs, 5S ribosomal Proteomics and ribosome profiling revealed that the deregulation
phenotype. RNA, U6 small nuclear RNA, vault RNA of these two tRNAIle isoacceptors resulted in increased translation of
and other small RNAs. growth-promoting mRNAs enriched in the AUA codon, which is cognate
Lineage-negative to the upregulated tRNAIleUAU (Fig. 2). Of note, the dually modulated cells
haematopoietic stem tRNA misacylation had a distinct proliferation advantage relative to control cells under
and progenitor cells The charging of a transfer RNA hypoxia and oxidative stress: two key features of the metastatic tumour
Immature blood cells that lack with the wrong amino acid, not the environment. Moreover, repression of tRNAIleGAU and upregulation of
surface protein markers found on amino acid cognate to the anticodon tRNAIleUAU have been observed in human breast tumours that had higher
mature blood cells. of the transfer RNA. rates of metastatic relapse32.
Several tumour suppressor and pro-tumorigenic transcription
Rare codons Wobble pairing factors have been reported to exert opposing effects on Pol III activity,
Codons found with the lowest Non-Watson–Crick base pairing of the although their effects on global tRNA pools versus specific isodecoder
frequency in genes compared with first anticodon nucleoside: inosine can genes have not been analysed in detail57,58. Of note, the tumour suppres-
synonymous codons in the same group. pair with adenosine, uridine or cytidine; sors p53 and pRb were both reported to bind to TFIIIIB and prevent
guanosine can pair with cytidine or recruitment of Pol III to its target genes59–61, whereas binding of the
Ribosome uridine; uridine can pair with adenosine MYC oncoprotein to TFIIIB stimulates the Pol III promoter62,63 (Fig. 3).
An essential ribonucleoprotein complex or guanosine. Moreover, Pol III activity is also stimulated by the mechanistic target
that performs protein synthesis in cells. of rapamycin (mTOR) and Ras oncogenic pathways64. Despite ChIP–
seq data showing overlap of several other transcription factors with
Pol III sites19,65, functional evidence of a transcription factor directly
although with much later latency than the tRNASer(Ala)-expressing cells binding upstream of a subset of tRNA genes has only been recently
(Fig. 3). Interestingly, the misincorporation of serine at alanine codons provided41. The authors found that overexpression of the transcrip-
did not impact cell viability in vitro but did provide NIH-3T3 cells with tion factor SRY-box transcription factor 4 (SOX4) in glioblastoma cells
an advantage during tumour growth in vivo54. The contribution of caused cell cycle exit41, in line with previous work showing that SOX4
particular tRNA variants to cancer has not been formally investigated expression can contribute to the reprogramming of glioblastoma cells
thus far, especially as the effects from mutating one tRNA gene are to a more terminally differentiated state66,67. Surprisingly, ChIP–seq
expected to be alleviated by the redundant copies of the tRNA genes uncovered SOX4 binding to over 200 tRNA gene loci in glioblastoma
in the same isoacceptor family. Nonetheless, as the aforementioned cells and mechanistic analysis has found that SOX4 binding reduced
studies suggest, the effects of tRNA variants may become apparent the recruitment of TFIIIB and Pol III at tRNA promoters and repressed
via epistatic interactions with specific mutated cancer genomes and/or target tRNA genes. Using functional assays, the authors showed that
under stress conditions, such as that of the tumour microenvironment SOX4 bound to a motif upstream of the tRNA-iMet-CAT-1 isodecoder
or during metastatic progression. family and repressed transcription. However, SOX4 did not alter the
Mutations in the internal A and B boxes, as well as in the immedi- expression of the related tRNA-iMet-CAT-2 isodecoder, and ectopic
ate flanking regions of the tRNAs, may also alter the transcriptional overexpression of tRNA-iMet-CAT-2 partially rescued the total tRNAiMet
output of tRNA genes. It was already appreciated in the 1980s that levels and the proliferation defect induced by SOX4 overexpression41.
transcription of tRNA genes can be modulated by 5′ upstream flanking It is unclear whether all the putative tRNA genes targeted have similar
regions outside of the conserved internal promoter elements in various SOX4-binding motifs and whether they are all responsive to SOX4 bind-
organisms, including humans55,56. However, to our knowledge, recur- ing. Nevertheless, this work suggests that specific transcription factors
rent features or functional motifs in the flanking regions of tRNA genes may directly regulate particular tRNA genes during cancer progression
have not been comprehensively and systematically investigated so far. (Fig. 3). Interestingly, SOX4 has been shown to have opposing roles in
cancer progression, acting as a tumour suppressor in certain tumour
Altered Pol III activity deregulates tRNAs in cancer types while having pro-oncogenic and pro-metastatic roles in other
The transcriptional control of tRNA genes depends on Pol III, with Pol III cancer types68,69, which raises the question of whether the dual role
occupancy indicative of active tRNA expression. In contrast to yeast, of SOX4 in tumorigenesis is, at least in part, mediated via differential
where virtually all tRNA genes are expressed, chromatin immunopre- modulation of tRNA expression in different cell types.
cipitation coupled with sequencing (ChIP–seq) experiments have found In addition to transcription factors, Pol III activity at tRNA genes is
that Pol III only occupies approximately 50% of all predicted human also influenced by chromatin accessibility19,20,58. DNA methylation has
tRNA genes. Interestingly, even though a set of approximately 120 tRNA been found to repress tRNA gene transcription in vitro70 and inhibit

Nature Reviews Cancer

Review article
TFIIIC binding to DNA71. Interestingly, DNA methylation has been sug-
gested to have a role in regulating the levels of specific tRNAs in cancer72.
Mining DNA methylation data from The Cancer Genome Atlas revealed
that 60 of the 71 tRNA genes with at least one CpG island covered by
the methylation array that was used exhibited statistically significant
differences between normal and tumour samples. In addition, 86 cases
were reported for which the methylation status of a tRNA gene was
associated with significant differences in overall patient survival, with
certain tRNA genes showing an effect in more than one cancer type.
Functional assays have revealed that the expression of two isodecoder
genes, tRNA-Arg-TCT-4-1 and tRNA-Ile-AAT-8-1, was indeed regulated
by DNA methylation. Moreover, unmethylated tRNA-Arg-TCT-4-1 has
been shown to be associated with significantly worse overall survival
outcome of patients with kidney papillary cell carcinoma and uterine
corpus endometrial carcinoma72. These findings suggest that the methy­
lation of tRNA genes may be an important mechanism for controlling
specific tRNA gene expression in cancer progression (Fig. 3).
Altered tRNA stability in cancer
tRNA levels can also be regulated post-transcriptionally through
changes in tRNA processing and stability. Mutations in tRNA processing
factors associated with alterations in tRNA stability have been observed
in several non-malignant human disorders7; however, such mutations
have only rarely been implicated in cancer progression, perhaps
because they would probably result in global reduction of tRNA levels,
impaired protein synthesis and growth inhibition. Thus far, only muta-
tions in the gene elaC ribonuclease Z 2 (ELAC2), which encodes the long
form of RNase Z in eukaryotes, have been associated with cancer73 and
may be low-penetrance susceptibility markers of prostate cancer74.
Nucleobase modifications are also an important determinant of
tRNA stability. Modifications outside the anticodon arm are linked to
stabilizing tRNA structure and inhibiting tRNA degradation, whereas
modifications in the anticodon arm generally regulate tRNA decoding
function24. The degradation of hypomodified tRNAs has mostly been
studied in Saccharomyces cerevisiae, in which two exonucleolytic path-
ways that mediate the breakdown of hypomodified tRNAs have been
described. The Trf4–Air2–Mtr4p polyadenylation (TRAMP) complex,
the members of which are conserved in humans75, degrades tRNAiMet
that lacks N1-methyladenosine at position 58 in a 3′→5′ direction76.
Hypomodified and unstable tRNAs are also degraded 5′→3′ by a dedi-
cated, rapid tRNA decay pathway in different yeasts77–79, with circum-
stantial evidence of it being conserved across eukaryotes, including in
humans80. One of the tRNA body modifications necessary to prevent
tRNA degradation in S. cerevisiae is N7-methylguanosine (m7G) at posi-
tion 46 (ref. 78). Interestingly, the RNA m7G methyltransferase complex,
comprising methyltransferase-like 1 (METTL1) and WD repeat domain 4
(WDR4), is overexpressed in various cancers81–85. Overexpression
of wild-type but not catalytically inactive METTL1 promoted malig-
nant phenotypes in in vitro assays and increased m7G modification
on a subset of tRNAs in various cell lines, including mouse embry-
onic fibroblasts81, intrahepatic cholangiocarcinoma (ICC)84, hepato-
cellular carcinoma (HCC)85, oesophageal squamous cell carcinoma
(ESCC)82, and head and neck squamous cell carcinoma (HNSCC)83
cell lines. A conditional Mettl1 knock-in mouse showed in vivo that
increased METTL1 expression promoted ESCC82, HCC85 and HNSCC83
tumorigenesis. Conversely, depletion of METTL1 or WDR4 inhibited
tumour growth in mouse xenograft assays using various cancer lines,
including acute myeloid leukaemia, glioblastoma, liposarcoma81,
ICC cells84, HCC cells85, ESCC cells82 and HNSCC cells83. A conditional
Nature Reviews Cancer
Mettl1-knockout mouse model revealed that, consistent with the xeno-
graft assays, loss of Mettl1 inhibited HCC85, ESCC82 and HNSCC83 tumo-
rigenesis. Mechanistically, depletion of either METTL1 or WDR4 caused
hypomodification of specific tRNAs and reduced the overall levels of a
subset of m7G-modified tRNAs in glioblastoma cells81 and ICC84, ESCC82,
HCC85 and HNSCC83 cell lines. Moreover, altering METTL1–WDR4
levels changed the expression of genes related to cell cycle and acti-
vated oncogenic pathways81–85. Some of these studies81,82,84,85 have
provided some analyses to show that the METTL1–WDR4 effects on
tumour growth are mediated, at least partially, via their downstream
tRNA targets. For example, overexpressing tRNAArgUCU recapitulated
the pro-tumorigenic effects and proteome changes induced by
METTL1–WDR4 ectopic expression81. Interestingly, overexpression
of tRNAArgUCU in simian virus 40 T antigen-immortalized mouse embry-
onic fibroblasts increased their anchorage-independent growth in
soft agar compared with control cells. Moreover, ectopic expression
of tRNAArgUCU in primary non-leukaemic NrasG12D/+ lineage-negative
haematopoietic stem and progenitor cells promoted colony formation
during serial replating assays relative to controls, providing in vitro evi-
dence for a pro-tumorigenic role for this isoacceptor81 (Fig. 2). However,
in the absence of exhaustive epistatic interaction analyses between
METTL1–WDR4 and its target tRNA genes in each studied cancer type,
it is challenging to draw conclusions about which tRNAs mediate the
complex effects of the methyltransferase complex on oncogenesis.
Moreover, METTL1, as well as most tRNA-modifying enzymes, can act
on other RNA species, such as mRNAs and microRNAs (miRNAs), apart
from tRNAs24,86,87. It would be important to disentangle the potential
effects of METTL1 depletion on tRNAs versus other RNA species in
future studies, as well as to biochemically assess whether tRNAs lacking
m7G are subjected to increased degradation in human cells. Neverthe-
less, these studies provide support for exonuclease-mediated decay
pathways regulating tRNA levels in cancer (Fig. 3) and motivate the
characterization of the molecular details of such pathways.
Finally, experimental modulation of tRNA levels using small
hairpin RNAs suggested that RNAi pathways could regulate tRNA
expression21,88. A recent study has provided, to our knowledge, the
first evidence of naturally occurring miRNA-mediated regulation of
tRNAs42. The authors found that unmodified pre-tRNAiMet is a target
of the tumour suppressor miR-34a using in vitro pulldowns and the
Argonaute 2 (AGO2) cleavage assay. The authors provided further
in vivo evidence of RNAi-mediated control of tRNAiMet levels by modulat-
ing miR-34a levels and by using small hairpin RNAs and small interfering
RNAs targeting tRNAiMet in breast cancer cells. Limited epistasis experi-
ments have suggested that the miR-34a cellular phenotypes were
partially mediated through modulation of tRNAiMet levels42.
Microenvironmental influences on tRNAs in cancer
The excessive proliferation of cancer cells causes the centre of solid
tumours to become deprived of nutrients and oxygen. This exposes
cancer cells to environmental stresses, which have been shown to
regulate tRNAs in yeast14. Some of the most prominent stresses that
cancer cells encounter are hypoxia and amino acid deprivation39.
Hypoxia can increase reactive oxygen species production89,90 and thus
oxidative stress. The effects of reactive oxygen species on tRNAs are
multifaceted91. In brief, oxidative stress can change the modification
patterns of tRNAs, affecting their decoding abilities91–93. Increased
levels of reactive oxygen species also affect tRNAs in non-canonical
ways through increased mischarging of non-Met-tRNAs with Met94,
selective retrograde transport of tRNAs to the nucleus95 and tRNA
Review article
fragmentation96–99. These tRNA-related response pathways are believed
to help protect the cells from the deleterious effects of oxidative stress
and raise the question of whether they are altered in cancer cells as a
means for adapting to hypoxic conditions.
Amino acid deprivation can induce an accumulation of uncharged
tRNAs that bind to and activate the protein kinase general control
non-depressible 2 (GCN2). GCN2 is an essential kinase in the integrated
stress response, a short-lived pathway that is necessary for maintain-
ing homeostasis or enabling apoptosis during prolonged or excessive
exposure to cellular stress. GCN2 phosphorylates eukaryotic initiation
factor 2 (eIF2) on its α-subunit at Ser51, causing global inhibition of
protein synthesis and selective upregulation of the translation of acti-
vating transcription factor 4 (ATF4), which in turn induces expression
of adaptive stress response genes36,100. Another, largely underappreci-
ated consequence of amino acid starvation in cancer was described
to be tRNA misacylation (Fig. 3). The cytokine IFNγ has been found to
trigger the depletion of the essential amino acid tryptophan (W), which
in turn caused tryptophanyl-tRNA synthetase 1 (WARS1) to erroneously
mischarge tRNATrp with phenylalanine (F)101. The authors referred to the
W > F containing peptides as ‘substitutants’, as they are not genetically
encoded, and used functional reporter assays and large-scale proteom-
ics to reveal their existence in multiple cancer types. Interestingly, the
W > F substitutants were enriched in tumours compared with matched
adjacent normal tissues, and they could diversify the antigens pre-
sented by HLA class I at the cell surface of cancer cells to activate T cell
responses101. This study highlights the effects of short-term amino acid
starvation on the cancer proteome.
In a recent study, long-term effects of specific amino acid pertur-
bations in cancer were analysed. Computational analysis of tumour
genome sequencing data has revealed that human cancers are enriched
in codon-switching events that result in a loss of arginine codons,
especially in colorectal and stomach cancers102. Limiting arginine
availability in vitro to physiologically low levels found in such human
tumours caused several colorectal cancer cell lines to mutate arginine
codons to other codons, thereby ‘losing’ arginine codons. A simi-
lar arginine codon-switching evolution has also been demonstrated
in vivo as patient-derived colorectal cancer xenografts that underwent
several rounds of liver metastasis selection had significantly more
arginine codon-switching mutations than the parental tumours. The
authors note that the liver is a common site of metastasis for colorec-
tal and gastric cancers, and that it has a high expression of arginase,
the arginine-degrading enzyme. Mechanistically, arginine restriction
both perturbed nucleotide synthesis and caused an acute depletion
of tRNAArg. This caused ribosome stalling at select arginine codons
and a proteomic shift from arginine-rich to arginine-low proteins102.
Interestingly, the acute degradation of tRNAArg seems to be partially due
to loss of aminoacylation (Fig. 3), as depleting arginyl-tRNA synthetase 1
(RARS1) reduced tRNAArg levels but no other tRNA species102, in line
with previous work showing that depletion of leucyl-tRNA synthetase 1
(LARS1) reduced the charging of certain leucyl-tRNAs and their overall
mature tRNA levels103. The pathway responsible for the degradation
of the uncharged tRNAs requires further study, as do the downstream
pathways engaged in this response.
Roles of aminoacyl-tRNA synthetases in cancer
aaRSs are evolutionarily ancient enzymes that charge tRNAs with
their cognate amino acids and are thus central to the canonical
function of tRNAs in protein synthesis. Owing to their essential
roles in translation, mutations in aaRSs are associated with various
Nature Reviews Cancer
developmental disorders104. Although aaRSs have functions not related
to protein synthesis that have been implicated in cancer (reviewed in
refs. 105–108), here we detail the involvement of aaRSs in cancer based
on their canonical protein synthesis functions.
To sustain unchecked proliferation, cancer cells evolve to alter
translational output by both increasing overall protein synthesis and
modulating specific mRNA programs38,109. Consistent with this, an
unbiased analysis of The Cancer Genome Atlas data found that different
cancers exhibited distinct patterns of aaRS expression110. In a recent
study, LARS1 levels were found to be reduced in breast cancer tumours
and cell lines compared with normal tissues and untransformed mam-
mary epithelial cells103. Moreover, LARS1 levels became repressed
during the oncogene-mediated transformation of normal mammary
epithelial cells. Using a genetic mouse model of breast cancer, mono-
allelic deletion of Lars1 in mouse mammary glands enhanced tumour
formation and proliferation. Mechanistically, LARS1 depletion caused
downregulation of select leucine tRNAs. Lower tRNALeu levels inhibited
protein synthesis in a codon-dependent manner, which resulted in
repression of multiple growth-suppressive genes, including epithelial
membrane protein 3 (EMP3) and γ-glutamyltransferase 5 (GGT5)103.
Intriguingly, this study has also revealed that specific isoacceptors
may act as suppressors of oncogenic transformation, as certain leucine
tRNAs were observed to become downregulated in breast cancer cells
compared with untransformed human breast epithelial MCF10A
cells. Moreover, depletion of tRNALeuCAG has been found to enhance the
soft agar colony formation of oncogene-transformed MCF10A cells
relative to controls103 (Fig. 2). Another recent study has also described a
canonical role for an aaRS in cancer. The authors found that valyl-tRNA
synthetase 1 (VARS1) became upregulated by NOTCH1 signalling in
T cell acute lymphoblastic leukaemia. Depletion of VARS1 impaired
the translation of mRNAs encoding mitochondrial electron transport
chain complex I subunits, resulting in reduced complex I formation,
impaired mitochondrial activity and reduced cellular fitness111. Over-
all, these studies have suggested that aaRSs can become modulated
in a cancer context-dependent manner and selectively modulate
codon-dependent translational gene networks during tumorigenesis.
Functions of tRNA fragments in cancer
An active area of research that has flourished in recent years relates to
the non-canonical roles of tRNA-derived fragments (tRFs; also referred
to as tRNA-derived small RNAs). tRFs are small, single-stranded RNA
molecules derived from mature or precursor forms of tRNAs112 that
were first detected in the urine of patients with cancer in the 1970s113,114.
Initially considered inconsequential products of degradation, tRFs
are now established as bona fide biologically active small non-coding
RNAs implicated in various biological processes112. High-throughput
sequencing of small RNAs has afforded the identification of tRFs
across all domains of life115 and the assembly of databases curating
tRFs identified in next-generation sequencing cancer data116,117.
Depending on their biogenesis pathways, tRFs can be broadly
classified into six distinct classes: 5′ or 3′ tRNA halves (tiRNA), tRF-1,
tRF-5, tRF-3 and internal tRFs (i-tRFs)112. The tRNA halves were the first
tRFs characterized in a model organism118 and are generally associated
with responses to various cellular stresses such as oxidative stress and
amino acid starvation119. The tRNA halves are generated by cleavage
within the anticodon loop by angiogenin99,120, RNase L121 or RNase 1
(ref. 122). tRF-1s are generated during tRNA maturation from the
3′ trailer sequences of pre-tRNAs following cleavage by RNase Z123.
The mechanism underlying the generation of the remaining classes of
Review article

tRFs is still largely unknown112. The functions and modes of action
of tRFs are very diverse and have been reviewed elsewhere112,124–126.
Advancements in small-RNA sequencing and computational biol-
ogy tools have enabled efforts aimed at identifying specific tRFs or
tRF signatures that could be used as biomarkers for various cancers
(Table 2). Here we summarize studies that have used functional assays
to characterize tRF effects on cancer progression and/or their molecu-
lar mechanisms of action. On a technical note, tRFs are lowly abundant
compared with their parental tRNAs127 and thus specifically deplet-
ing tRFs generally minimally impacts the levels of the parental tRNA
molecules that they arise from. Nevertheless, care should be taken
in interpreting studies that do not control for the mature tRNA levels
and/or lack additional mechanistic insights into the tRF molecular
function.
Tumour-suppressive tRFs
Several tRFs have been identified in various cancers as tumour sup-
pressive, often interacting with Argonaute proteins to engage the RNAi
machinery and inhibit the translation of target mRNAs (Fig. 4a). CU1276,
a tRF-3 derived from tRNAGlyGCC, has been observed to be downregulated
in B cell lymphoma cells and DLBCL and found to target the replication
protein A1 (RPA1) mRNA that encodes an essential DNA replication and
DNA repair protein128. Ts-3676 has been reported to be downregulated
in samples from patients with chronic lymphocytic leukaemia and has
been found to target the 3′ untranslated region of the TCL1A oncogene
mRNA, reducing its translation. Interestingly, even though ts-3676
interacted with AGO1 and AGO2, it also interacted with Piwi-like pro-
tein 2 (PIWIL2), suggesting that it might control gene expression
through direct site-specific DNA methylation via the PIWI pathway129,130.

Table 2 | Studies on tRNA-derived fragments as cancer biomarkers

Cancer type Identity of tRFs Sample sizes Sample type Ref.

Bladder cancer tRF-5: 5′-tRF Lys

CTT Discovery: 230 bladder cancers Tissue biopsies 154
Validation: 412 TCGA bladder cancers
Breast cancer tRF-1: tRF-Arg-CCT-017, tRF-Gly-CCC-001 Discovery: 8 breast cancers versus 4 controls Plasma samples 155
tRNA half: tiRNAPheGAA003 Validation: 120 breast cancers versus 112 controls
Thirty differentially expressed tRFs (5′ halves; 16 paired breast cancers and normal tissues (6 for initial Tissue biopsies 156
tRF-5, tRF-3, i-tRF) discovery, 10 for validation)
Trastuzumab-resistant i-tRFs from tRNACysGCA: tRF-30-JZOYJE22RR33 57 HER2+ breast cancers previously treated with Serum samples 157
HER2+ breast cancer and tRF-27-ZDXPHO53KSN trastuzumab
Chronic lymphocytic tRF-1, i-tRF, 5′-tRF 20 patients with chronic lymphocytic leukaemia Leukaemia or blood 158
leukaemia and 6 healthy donors samples
Clear cell renal cell 5′-tiRNA4ValAAC Discovery: 18 paired clear cell renal cell carcinomas Tissue biopsies 159
carcinoma and normal tissues
Validation: 118 clear cell renal cell carcinomas and
74 normal tissues
Colorectal cancer 5′-tRFGlyGCC (tRF-35-PNR8YP9LON4VN1) Discovery: 3 colorectal cancers versus 3 healthy controls Plasma samples 160
Validation: 105 colorectal cancers and 90 healthy controls
tRF-3s: tRF-22-WB86Q3P92 (from tRNAThrAGT), Discovery: 104 paired colorectal cancers and normal Tissue biopsies 161
tRF-22-WE8SPOX52 (from tRNAGlyGCC), tissues
tRF-22-WE8S68L52 (from tRNAGlyCCC), Validation: TCGA data, 603 primary tumours and
tRF-18-8R1546D2 (from tRNAAlaAGC) 11 adjacent normal tissues
Non-small-cell lung tRNA halves, potentially other Discovery: 30 pairs of non-small cell lung cancers and Tissue biopsies, 162
cancer normal tissues serum samples
Validation: 60 pairs of non-small cell lung cancers and
normal tissues; 167 serum samples (34 normal and 133
non-small-cell lung cancers)
tRF-5: tRF-31-79MP9P9NH57SD 96 non-small-cell lung cancer, 96 healthy controls and Serum samples 163
(from tRNAValAAC/CAC) 20 pairs of non-small-cell lung cancer serum samples
pre-surgery and post-surgery
Gastric cancer i-tRF: tRF-31-U5YKFN8DYDZDD Discovery: 3 paired gastric cancer samples Tissue biopsies, 164
(from tRNAValTAC) Validation: 24 pairs of gastric cancer samples; sera from serum samples
111 patients with gastric cancer, 48 gastritis and 89 healthy
controls; 28 pre-operative and post-operative patients with
gastric cancer
Ovarian cancer i-tRF-Gly-GCC Screening: 98 ovarian cancers Tissue biopsies 165
Validation: 100 ovarian cancers
Prostate cancer All tRF classes; ratio of tRF-315 (from Discovery: 31 prostate cancer and 8 non-malignant Tissue biopsies 166
tRNALysCTT) to tRF-544 (from tRNAPheGAA) samples
proposed as a biomarker Validation: 65 prostate cancers (cohort 1) and 104 prostate
cancers (cohort 2)
i-tRF, internal transfer RNA-derived fragment; TGCA, The Cancer Genome Atlas; tRNA, transfer RNA.

Nature Reviews Cancer

Review article

a 3′ b c mTOGs tRF-21
3′ YBX1 YBX1
5′
tRF
tRF-Glu-YUC tRF-Asp-GUC
PAIP1 DDX17
mRNA mRNA YBX1 YBX1 PABPC1 hnRNPL
AGO target degradation S52
tRF-Gly-UCC tRF-Tyr-GUA

Tumour-suppressive tRFs
Destabilization of growth- PAIP1 hnRNPL
tRFs mRNAs PABPC1 DDX17
promoting mRNAs

CU1276 RPA1
ts-3676 TCL1A
tRF-24-V29K9UV3IU GPR78 Translation of Altered splicing
5′ PES mRNAs CASP9, MACROH2A1
Oncogenic or tumorigenic tRFs
tRF-3017A NELL2
tRF-3019a FBX047 Breast cancer metastasis MDS progression to AML Pancreatic cancer progression

d e NCL NCL
RBM17
5′-tiRNA Gly
GCC
UHM 5′-tRF
Cys
5′-tRFCys
5′ Oligomerization Breast
RB

cancer
5′
M

RBM17 metastasis
17

Altered
splicing Thyroid 5′ mRNAs 3′ 3′
3′ 3′
cancer 3′ 3′
Target mRNAs
Spliceosome progression
PAFAH1B1 and MTHFD1L mRNA stabilization

Fig. 4 | Examples of oncogenic and tumour-suppressive transfer RNA-derived
fragments in cancer. a, Transfer RNA (tRNA)-derived fragments (tRFs) can
engage the cellular RNA interference machinery and inhibit the translation
of target messenger RNA (mRNA) by inducing mRNA degradation. Potential
tumour-suppressive and tumorigenic microRNA-like tRFs and their target genes
are shown. b, tRFs can bind to RNA-binding proteins (RBPs), such as the Y-box
binding protein 1 (YBX1), and segregate them away from their target mRNAs,
which, for example, results in the destabilization of growth-promoting target
mRNAs and suppression of breast cancer metastasis. c–e, tRFs can modulate the
interactions of RBPs with other proteins. Examples of tumour-suppressive tRFs
modulating protein–protein interactions include pseudouridylated mini tRNAs
containing a 5′ terminal oligoguanine (mTOGs, a type of tRF-5s) that inhibit the
binding of poly(A)-binding protein 1 (PABPC1) to the translational co-activator
poly(A)-binding protein interacting protein 1 (PAIP1), which represses
the translation of mRNAs containing pyrimidine (Y)-enriched sequences
(PES) in their 5′ untranslated regions and thus could inhibit progression of
myelodysplastic syndrome (MDS) to acute myeloid leukaemia (AML) (left).
In pancreatic cancer (right), tRF-21-VBY9PYKHD binds to Ser52 on heterogeneous
nuclear ribonucleoprotein L (hnRNPL) and prevents its phosphorylation at
that residue, which inhibits the formation of an RNA splicing complex with
DEAD-box helicase 17 (DDX17). This results in altered splicing of select
mRNAs (part c). Examples of tumorigenic tRFs modulating protein–protein
interactions (parts d and e). The 5′-tiRNAGlyGCC may promote thyroid cancer
progression through altered splicing (part d) and 5′-tRFCys may promote
breast cancer metastasis through mRNA stabilization (part e). AGO,
Argonaute; CASP9, caspase 9; MACROH2A1, macroH2A.1 histone; MTHFD1L,
methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like; NCL,
nucleolin; PAFAH1B1, platelet-activating factor acetylhydrolase 1b regulatory
subunit 1; RBM17, RNA-binding motif protein 17; UHM, U2AF homology motif.

Finally, the knockdown of tRF-24-V29K9UV3IU, a tRF-5 downregulated
in gastric cancer tumours, promoted the growth and metastasis of
tumour xenografts of MKN-45 gastric cancer cells, whereas overexpres-
sion of the tRF inhibited cell proliferation, migration and invasion131. The
authors found that tRF-24-V29K9UV3IU targeted, in a miRNA-like man-
ner, the G protein-coupled receptor GPR78, which had previously been
shown to promote malignant phenotypes in lung cancer132. Epistasis
experiments have shown that altered GPR78 levels accounted partially
for the phenotypes caused by overexpression of tRF-24-V29K9UV3IU131.
Another major mode of action for tRFs in cancer involves interac-
tions with RNA-binding proteins (RBPs). RBPs regulate gene expression
post-transcriptionally through a diverse set of mechanisms, such as
modulating the translation of specific mRNAs133. A tRF can bind to an
RBP and competitively inhibit the binding of the RBP to its mRNA tar-
gets, affecting the stability of the respective mRNAs (Fig. 4b). Interest-
ingly, hypoxia has been found to induce specific tRNA fragmentation in
breast cancer cells that was functionally linked to metastatic potential97
(Fig. 3). In highly metastatic breast cancer cells, the hypoxic induction
of a subset of tRFs was blunted, and the group of four tRFs derived
from tRNAGluYTC, tRNAAspGTC, tRNAGlyTCC and tRNATyrGTA was repressed in
highly metastatic cells relative to poorly metastatic cells97. These tRFs
inhibited metastasis when upregulated and, conversely, promoted
metastasis when inhibited in breast cancer cells. Mechanistically,
the tRFs derived from tRNAGluYTC, tRNAAspGTC, tRNAGlyTCC and tRNATyrGTA
bound to and competitively inhibited the binding of Y-box binding
protein 1 (YBX1) to a set of growth-promoting and oncogenic mRNAs.
Because YBX1 stabilized those transcripts, tRF binding to YBX1 led
to their destabilization and suppressed metastasis97. Another study
has also demonstrated that fragmentation of tRNAGlu can give rise to
a tumour-suppressive tRF in breast cancer. The tRF identified in this
study, termed tRF3E, was shown to bind to nucleolin and was suspected
of inhibiting its binding to its downstream target mRNAs134.
tRFs can also inhibit the interaction of RBPs with other proteins
(Fig. 4c). Recently, a class of pseudouridylated tRF-5s called mini tRNAs

Nature Reviews Cancer

Review article
containing a 5′ terminal oligoguanine (mTOGs) have been shown to
regulate translation initiation, implicating them in myelodysplastic
syndrome progression135. Pseudouridylated mTOGs have been found
to bind to poly(A)-binding protein 1 (PABPC1) and prevent its interac-
tion with translational co-activator poly(A)-binding protein interacting
protein 1 (PAIP1), which resulted in repression of mRNA transcripts con-
taining pyrimidine-enriched sequences in their 5′ untranslated regions.
This translational repression prevented aberrant protein synthesis that
could drive myelodysplastic syndrome progression to acute myeloid leu-
kaemia, although the identity of the repressed oncogenic translational
programs has not yet been determined135,136. Finally, in pancreatic cancer,
an i-tRF derived from tRNAGlyGCC, tRF-21-VBY9PYKHD, has been found
to be downregulated in tumours compared with normal tissues and in
later-stage disease versus earlier stages137. Extensive gain-of-function
and loss-of-function experiments in vivo and in vitro have supported the
conclusion that tRF-21-VBY9PYKHD is a tumour suppressor in pancreatic
cancer. The authors showed that tRF-21-VBY9PYKHD interacted with
heterogeneous nuclear ribonucleoprotein L (hnRNPL) at residue Ser52,
which blocked phosphorylation of hnRNPL, inhibiting its formation of an
RNA splicing complex with DEAD-box helicase 17 (DDX17). This resulted
in altered splicing of mRNAs encoding caspase 9 and macroH2A.1 his-
tone, inhibiting the formation of the anti-apoptotic caspase 9b and the
pro-invasive mH2A.1.2 isoforms. The authors also showed that tRF-21 for-
mation is mediated by serine and arginine-rich splicing factor 5 (SRSF5),
which is downregulated following treatment with the inflammatory
cytokines LIF IL-6 family cytokine (LIF) or IL-6 (ref. 137). These studies
have revealed that tRF binding to RBPs and altering their interactions is
a major mechanism of action for this class of non-coding RNAs.
Tumour-promoting tRFs
Several tRFs can downregulate the expression of potential tumour sup-
pressors. In gastric cancer, tRF-3017A and tRF-3019a have been found to
be upregulated in tumours138,139. Experimentally, modulating the levels
of each of the tRFs impacted cell migration and invasion in vitro138,139.
The authors then showed that tRF-3017A targeted neural EGFL-like 2
(NELL2)138, whereas tRF-3019a targeted F-box protein 47 (FBXO47)139,
and suggested that repression of these potential tumour suppressors
mediated the phenotypes of these tRFs, respectively (Fig. 4a).
Oncogenic tRFs can also mediate their effects by regulating
RBPs (Fig. 4d,e). For example, the 5′ tRNA half 5′-tiRNAGlyGCC has been
found to be overexpressed in papillary thyroid carcinoma (PTC)140.
Exogenous expression of 5′-tiRNAGlyGCC increased cell proliferation
and cell migration in vitro in PTC cells, whereas its depletion had
the opposite effects. Consistent with the in vitro data, knockdown
of 5′-tiRNAGlyGCC inhibited tumour growth in xenograft assays using
a PTC cell line140. Mechanistically, 5′-tiRNAGlyGCC bound the splicing
protein RNA-binding motif protein 17 (RBM17) via its U2AF homo­
logy motif domain, resulting in RBM17 stabilization and its translo-
cation to the nucleus. Overexpression of 5′-tiRNAGlyGCC in a PTC cell
line changed the splicing patterns of genes associated with onco-
genic signatures, indicating that 5′-tiRNAGlyGCC exerts its function in
part by RBM17-mediated alternative splicing140. Another tRF that
exerts its function via RBPs is the 5′-tRFCysGCA, which is upregulated
during breast cancer progression and associated with reduced sur-
vival outcomes in patients with breast cancer141. Depleting 5′-tRFCysGCA
inhibited breast cancer cell survival and metastasis in xenograft
models. Mechanistically, 5′-tRFCysGCA bound to nucleolin, leading
to its oligomerization and stabilization of mRNAs bound to nucleolin,
such as platelet activating factor acetylhydrolase 1b regulatory subunit 1
Nature Reviews Cancer
(Pafah1b1) and methylenetetrahydrofolate dehydrogenase (NADP+
dependent) 1-like (Mthfd1l). Further experiments confirmed that
both metabolic enzymes act downstream of 5′-tRFCysGCA to mediate its
pro-survival and pro-metastatic activities. Overall, this study revealed
that a tRF can act in a gain-of-function manner to promote the forma-
tion of a stabilizing ribonucleoprotein complex on mRNAs, resulting
in pro-tumorigenic phenotypes141.
Conclusions and future perspectives
The past 15 years have brought tRNAs into focus as dynamic and critical
regulators of cancer progression. Even though imperfect (Box 1), tech-
nological advances have uncovered global tRNA alterations in tumours
that suggest cancer cells optimize their tRNA pools to match the codon
usage of their transcriptomes, allowing for efficient translation. Fur-
thermore, studies have shown that individual tRNAs can have causal
roles in driving cancer progression via modulation of distinct gene
expression networks in a codon-dependent manner. These findings
open up new questions such as whether the tRNA abundance changes
during cancer progression disproportionally affect the translation effi-
ciency of genes enriched in rare codons, especially given the evidence
that the enrichment of rare codons in the common oncogene KRAS
influence its expression142, oncogenesis143,144 and potentially treatment
responses145. Moreover, the reports so far have only been restricted
to a handful of cancer types, indicating that the systematic analyses
of tRNA levels using dedicated tRNA profiling techniques in different
cancer types are needed (Box 1). Gain-of-function and loss-of-function
experiments are further needed to then pinpoint the tRNA changes that
functionally impact cancer progression in different cancer types (Box 1).
The tRNA-related basic research discoveries open new avenues
for diagnostic and therapeutic interventions for patients with can-
cer. For example, there is increasing interest in using small-molecule
inhibitors to regulate the activity of aaRSs in human disease, including
cancer146–148. Moreover, tRNAs themselves could be delivered as thera-
pies, as illustrated by recent proof-of-concept studies. For example,
transgenic overexpression of tRNAGly rescued pathogenic phenotypes
in Drosophila and murine models of Charcot–Marie–Tooth peripheral
neuropathy149. Other studies have illustrated different methods to
deliver tRNAs in mice, focusing on suppressor tRNAs150,151. A suppres-
sor tRNA carries a mutation in its anticodon that allows it to bind to a
stop codon and incorporate an amino acid in its place, which enables
readthrough of a premature stop codon introduced by a nonsense
mutation152. Using an adeno-associated virus system to deliver a sup-
pressor tRNATyr, readthrough of the mutant Idua gene (Idua(W401X);
TGG→TAG) was successfully induced and the pathogenic phenotypes
were partially rescued in a mouse model that recapitulates the human
lysosomal storage disease mucopolysaccharidosis type I150. Another
proof-of-concept paper used lipid nanoparticles to successfully deliver
functional suppressor tRNAs in mice151. An optimistic therapeutic
outlook inspired by these papers is the delivery of tRNAs to restore the
levels of tumour-suppressive tRNAs in human cancer.
The characterization of tRFs as bona fide functional non-coding
small RNAs coupled with sequencing of patient samples led to the
identification of many tRFs with functional roles in cancer. The isola-
tion and sequencing of small RNAs from liquid biopsies can aid the
discovery of tRF signatures that could serve as biomarkers indicative
of different cancer types and even cancer stages. Moreover, tRFs that
manifest their effects on cancer progression by regulating the activ-
ity of RBPs could be potential therapeutic targets using technologies
such as locked-nucleic acids153. Locked-nucleic acids could also be used
Review article
to target mature tRNAs that promote tumour growth or metastasis.
These are just a few of the potential translational applications of the
findings related to tRNA in cancer. We envision that even more appli-
cations and potential for therapeutic intervention will be discovered
as we continue to uncover how tRNA biology is functionally linked to
cancer progression.
Published online: xx xx xxxx
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Acknowledgements
The authors thank the members of the Tavazoie laboratory for critically reading the
manuscript and for their helpful suggestions and comments. The authors apologize to all
the scientists whose work could not be cited owing to space limitations. S.F.T. and A.M.P.
were supported by grants from the National Cancer Institute of the National Institutes of
Health under award numbers R01CA257153, R35CA274446 and U54CA261701, as well as
the Black Family Metastasis Center and the Breast Cancer Research Foundation. Molecular
graphics were performed with UCSF ChimeraX, developed by the Resource for Biocomputing,
Visualization and Informatics at the University of California, San Francisco, with support
from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and
Computational Biology, National Institute of Allergy and Infectious Diseases.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Reviews Cancer thanks Pierre Close and the other,
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