TRMT2A Is A Novel Cell Cycle Regulator Tha 2019 Biochemical and Biophysical
TRMT2A Is A Novel Cell Cycle Regulator Tha 2019 Biochemical and Biophysical
TRMT2A Is A Novel Cell Cycle Regulator Tha 2019 Biochemical and Biophysical
a r t i c l e i n f o a b s t r a c t
Article history: During the maturation of transfer RNA (tRNA), a variety of chemical modifications can be introduced at
Received 8 November 2018 specific nucleotide positions post-transcriptionally. 5-Methyluridine (m5U) is one of the most common
Accepted 16 November 2018 and conserved modifications from eubacteria to eukaryotes. Although TrmA protein in Escherichia coli
Available online 28 November 2018
and Trm2p protein in Saccharomyces cerevisiae, which are responsible for the 5-methylation of uracil at
position 54 (m5U54) on tRNA, are well characterized, the biological function of the U54 methylation
Keywords:
responsible enzyme in mammalian species remains largely unexplored. Here, we show that the
TRMT2A
mammalian tRNA methyltransferase 2 homolog A (TRMT2A) protein harbors an RNA recognition motif in
5-Methyluridine
Cell proliferation
the N-terminus and the conserved uracil-C5-methyltransferase domain of the TrmA family in the C-
Cell cycle terminus. TRMT2A predominantly localizes to the nucleus in HeLa cells. TRMT2A-overexpressing cells
display decreased cell proliferation and altered DNA content, while TRMT2A-deficient cells exhibit
increased growth. Thus, our results reveal the inhibitory role of TRMT2A on cell proliferation and cell
cycle control, providing evidence that TRMT2A is a candidate cell cycle regulator in mammals.
© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction tRNAs [1]. These modifications play essential roles in the fidelity of
translation, the folding and stability of tRNA [2e4] and the recog-
Transfer RNA (tRNA) is the key adaptor molecule responsible for nition of tRNA by the translation machinery [5].
the delivery of amino acids to the ribosome, where tRNA partici- One of the most abundant modifications found in tRNA is 5-
pates in the decoding of mRNA template into protein. Eukaryotic methyluridine (m5U or rT, ribothymidine). The ubiquitous pres-
cytoplasmic tRNA is transcribed by RNA polymerase III as a pre- ence of m5U at position 54 in the TjC stem-loop of eubacteria and
cursor molecule in the nucleus. Functional maturation of pre-tRNA in eukaryotic elongator tRNAs implies the pivotal role for this
involves a series of processing events, including numerous post- modification. In Escherichia coli, the enzyme that catalyzes the
transcriptional modifications. To date, more than 100 chemically formation of m5U54 is TrmA [6], while in Saccharomyces cerevisiae,
distinct post-transcriptional modifications have been reported for the enzyme responsible for this modification is Trm2p [7]. Both
TrmA and Trm2p belong to the S-adenosyl-methionine (SAM)-
dependent methyltransferase family, catalyzing methyl group
* Corresponding author. 1-1-1 Tennodai, Laboratory Animal Resource Center,
transfer from SAM to the C5 of U54 of tRNA [7e9]. Previous studies
University of Tsukuba, Tsukuba, Ibaraki, Japan. have shown that the presence of m5U54 increases the fidelity and
** Corresponding author. 1-1-1 Tennodai, Laboratory Animal Resource Center, efficiency of protein synthesis by stabilizing the three-dimensional
University of Tsukuba, Tsukuba, Ibaraki, Japan. structure of tRNA in vitro [10,11]. In mammals, TRMT2A and
E-mail addresses: akuno@md.tsukuba.ac.jp (A. Kuno), satoruta@md.tsukuba.ac.
TRMT2B are considered to be homologs of yeast Trm2p [12]. The
jp (S. Takahashi).
https://doi.org/10.1016/j.bbrc.2018.11.104
0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Y.-H. Chang et al. / Biochemical and Biophysical Research Communications 508 (2019) 410e415 411
predicted methyltransferase domain of TRMT2A shows a higher DNA staining with Hoechst 33342 (Molecular Probes) in Flur-
identity (36%) with that of Trm2p. Similar to Trm2p, TRMT2A har- omount medium (COSMO BIO). Slides were observed by Biorevo
bors a putative RNA recognition motif (RRM) in the N-terminus. By BZ-9000 fluorescence microscopy (Keyence), and images were ac-
contrast, the shared identity between TRMT2B and Trm2p is low quired using BZ-II Analyzer software (Keyence).
(28%), and the TRMT2B protein does not have an identifiable RRM
motif. This evidence indicates that TRMT2A is the likely mamma-
2.5. Cell proliferation and survival
lian homolog of Trm2p, for which the biological function remains
largely unexplored.
Cells were seeded in 24-well plates (2 104 per well) in tripli-
Here, we investigated the effects of TRMT2A on the growth in
cate. At 24, 48, 72, and 96 h post-plating, cells were trypsinized and
mammalian culture systems. TRMT2A-overexpressing HeLa cells
stained with trypan blue. Live cells with positive trypan blue
exhibit decreased cell growth and altered cell cycle profile, while
staining were counted using a hemocytometer. The cell survival
Trmt2a knock-out (KO) mouse embryonic fibroblasts (MEFs) show
rate was confirmed using a TC10 automated cell counter (Bio-Rad).
elevated cell growth. Our results provide evidence that TRMT2A
exerts an inhibitory effect on cell proliferation and is a promising
cell cycle regulator in mammals. 2.6. Clonogenic assay
2. Materials and methods Cells were seeded in 6-well plates (100 cells per well) and
cultured for 14 days. Colonies formed were fixed with 100% cold
2.1. Plasmids methanol for 30 min and then stained with 0.5% crystal violet (in
20% methanol) for 1 h. After air-drying, colony numbers were
The coding region of human TRMT2A was PCR-amplified from counted. Plate efficiency (PE) reflected the ratio of the number of
cDNA extracted from HeLa cells using the primers F 50 - colonies to the number of cells originally seeded.
CTGAATTCGATGAGTGAGAACCTCGACAAC-30 and R 50 -
CGGGATCCCTAGGATGAGGGGAAGGTCCCAG-3’. EcoRI and BamHI 2.7. Cell cycle analysis
restriction sites were added to the primers to facilitate directional
cloning into the pEGFP-c1 (BD Biosciences) vector for expression as Cells were seeded in 6-cm plates at 4 105 cells per dish. At 24 h
an EGFP-tagged fusion protein. Sequences were verified by Sanger after cell seeding, culture media were harvested to collect mitotic
sequencing using a BigDye™ Terminator v3.1 Cycle Sequencing Kit cells, which had rounded and were easily floating. Attached cells
(Applied Biosystems) and were analyzed on a 3500 Genetic were trypsinized and resuspended in 1 ml of complete DMEM
Analyzer sequencing machine (Applied Biosystems). Expression of culture medium. DNA was stained with 5 mM Vybrant DyeCycle
GFP-TRMT2A in HeLa cells was confirmed by immunoblotting and Violet (Invitrogen) at 37 C for 30 min. Cell cycle distribution ac-
immunofluorescence assay. cording to DNA contents was determined by CytoFLEX (Beckman
Coulter) and analyzed using CytoExpert software (Beckman
2.2. Transfection and generation of stable cell lines Coulter).
2.10. Statistics
3. Results
more than 95% by trypan blue staining in both cell lines (Fig. 3B). To
verify the inhibitory influence of TRMT2A on cell growth over a
longer time period and to monitor the growth capacity at the single
cell level, a clonogenic assay was performed. A plating efficiency of
approximately 80% was observed in GFP-HeLa control cells, while
only 60% of plated GFP-TRMT2A-HeLa cells formed colonies after 14
days of culturing (Fig. 3C). In addition, colonies of GFP-TRMT2A-
HeLa cells displayed a marked reduction in size (Fig. 3D),
implying that the increased expression of TRMT2A is adverse to cell
proliferation.
Fig. 4. Trmt2a KO MEFs exhibit increased cell proliferation. Trmt2a KO mouse embryonic fibroblasts (MEFs) were derived from KO mice. The knock-out efficiency was confirmed
at the (A) transcript and (B) protein levels. (C) WT and Trmt2a KO MEFs were seeded into 24-well plates in triplicate, and cell numbers were counted at 24, 48, 72, and 96 h in
culture. One representative result is shown from two independent experiments. **p < 0.01; ***p < 0.005. Differences were compared with the WT control.
unverified. Here, we showed that TRMT2A is the putative homolog in hepatomas than in normal liver tissue. However, the comparison
of the TrmA family and is a promising cell cycle-regulating protein. of tRNAs from two tumors could not provide evidence for a distinct
Overexpression of TRMT2A leads to reduced cell proliferation and correlation between degrees of tRNA modification and growth rates
an increase in the G2/M population, implying that increased and the histological characteristics of the tumors [31]. Another
TRMT2A expression is disadvantageous to cell growth and cell cycle study profiling modified nucleosides showed elevated m5U54
progression. The inhibitory effect of TRMT2A on cell proliferation is levels in the cell culture supernatants in MCF-7 breast cancer cells
confirmed by the observation in which Trmt2a KO MEFs exhibit compared with nontumorigenic MCF-10A mammary epithelial
faster cell growth than WT cells. Together, these results provide cells [32]. One of the reasons for the differences observed between
evidence for the conserved function of mammalian TRMT2A in cell these experiments may result from the origins of cancer and the
proliferation. In addition, we demonstrated that TRMT2A genes in cellular compartments where tRNA was extracted. Recent clinical
mammals are dispensable for survival since KO cells were viable. screenings have suggested overexpression of TRMT2A as a novel
Although the m5U54 is a universal modification in E. coli, the biomarker in HER2þ breast cancer patients, with a higher risk of
5
m U54 contents in eukaryotic tRNA vary. In mammals, including recurrence [33]. Of note, the TRMT2A expression pattern reported is
humans, many tRNAs have been reported to lack the 5- a cytoplasmic form. Since the U54 methylation was reported to
methyluridine at position 54, showing only approximately 50% of occur in the nucleus, and we observed a uniform nuclear localiza-
modified uridine in total tRNA [22,23]. For example, in the initiator tion of GFP-tagged TRMT2A, it is possible that cytoplasmic
tRNAMet
i , 5-methyluridine is completely replaced by adenosine, expression of TRMT2A is an aberrant form and perturbs the HER2
while in tRNAVal from mouse myeloma, unmethylated uridine is signaling pathway independent of its methyltransferase activity.
present instead of methylated uridine [24,25]. In some other tRNAs, Taken together, systematic analysis and comprehensive map-
including tRNAPhe, partial conversion of uridine to methyluridine ping of individual tRNA species and proteins altered by aberrant
has been revealed [26,27]. We also observed incomplete 5- m5U54 and TRMT2A are required. Understanding the role of
methyluridine modification because the m5U levels in HeLa cells TRMT2A-catalyzed m5U54 will uncover the target proteins and the
are much less methylated than control tRNAs extracted from E. coli underlying signaling pathways of TRMT2A in both physiological
by tRNA hydrophilic interaction liquid chromatography (HILIC) and pathological conditions.
analysis (methods modified from Ref. [28], data not shown). It is
interesting that partially methylated uridine in tRNAs purified from Conflicts of interest
human placenta and rat liver can be fully modified in vitro by an
excess amount of TrmA of E. coli [29]. Hence, we hypothesized that The authors declare no competing financial interests.
overexpression of TRMT2A in HeLa cells might lead to increased
levels of 5-methyluridine. However, the m5U levels in the entire Acknowledgments
pool of tRNAs extracted from GFP-TRMT2A-HeLa cells were meth-
ylated and comparable with those of GFP control cells, as demon- We would like to thank Masami Ojima for technical support and
strated by tRNA HILIC analysis (data not shown), implying that Keiko Amagai for the preparation of the Trmt2a KO MEFs. We also
increased amounts of TRMT2A do not affect the homeostasis of show appreciation to Dr. Mitsuyasu Kato (University of Tsukuba,
m5U modification in HeLa cells. On the other hand, our unpublished Japan) for the fruitful discussions. This work was supported by a
results demonstrated that 5-methyluridine levels in tRNAs extrac- Grant-in-Aid for Scientific Research (26221004) from the Ministry
ted from Trmt2a KO mice were completely lost. Hence, further of Education, Culture, Sports, Science, and Technology (MEXT) of
experiments to elucidate the specific tRNA targets of TRMT2A in Japan.
mammalian cells will be required to understand the significance of
5-methyluridine and TRMT2A.
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