RESEARCH ARTICLE

mTORC1-independent translation control in mammalian cellsby methionine adenosyltransferase 2A andS-adenosylmethionine
Received for publication, October 28, 2021, and in revised form, May 19, 2022 Published, Papers in Press, May 27, 2022,
https://doi.org/10.1016/j.jbc.2022.102084

Mahabub Alam1,2 , Hiroki Shima1 , Yoshitaka Matsuo3, Nguyen Chi Long1, Mitsuyo Matsumoto1 , Yusho Ishii1 ,Nichika Sato3, Takato Sugiyama4 , Risa Nobuta4 , Satoshi Hashimoto4 , Liang Liu1, Mika K. Kaneko5 ,Yukinari Kato5,6, Toshifumi Inada3, * , and Kazuhiko Igarashi1, *From the 1Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Department of Animal
Science and Nutrition, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University, Chattogram,
Bangladesh; 3Division of RNA and Gene Regulation, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
4
Laboratory of Gene Regulation, Department of Molecular Biopharmacy and Genetics, 5Department of Antibody Drug
Development, and 6Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan
Edited by Karin Musier-Forsyth

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyldonor for methylation of DNA, RNA, and proteins, SAM
levels affect gene expression by changing methylation patterns.
Expression of MAT2A, the catalytic subunit of isozyme MAT2,
is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced inproliferating cells and that RNA and protein components oftranslation machinery are methylated, we tested here whether
MAT2 and SAM are coupled with protein synthesis. By
measuring ongoing protein translation via puromycin labeling,
we revealed that MAT2A depletion or chemical inhibition
reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis,
indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany
reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysomebound RNA sequencing revealed that MAT2 inhibition
decreased translation efﬁciency of some fraction of mRNAs.
MAT2A was also found to interact with the proteins involved
in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally,
quantitative mass spectrometry revealed that some translation
factors were dynamically methylated in response to the activityof MAT2A. These observations suggest that cells possess an
mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system mayacclimate cells for survival when SAM synthesis is reduced,
whereas it may support proliferation when SAM is sufﬁcient.

* For correspondence: Kazuhiko Igarashi, igarashi@med.tohoku.ac.jp;
Toshifumi Inada, toshiinada@ims.u-tokyo.ac.jp.

Translation is the single largest energy expenditure sector ofcellular metabolism which uses around 20% of the total energyof cells (1–3). Since aberrant translation leads to dissipation ofenergy resources and various pathological consequences,translation is tightly regulated by multiple signaling pathways
in cells (4). Growth and proliferation of mammalian cells
require signal-mediated stimulations of translation initiated by
the availability of extracellular factors such as insulin and othergrowth factors, nutrition, endocrine secretions and metabolites. The proliferating cells integrate the presence of these
extracellular molecules to induce translation (5, 6). So far as
explored, the phosphoinositide 3-kinase–AKT and themitogen-activated protein kinase pathways are the main upstream controllers of translation in cells (7, 8). Both pathways
converge on mechanistic target of rapamycin complex 1(mTORC1) to regulate translation. mTORC1 phosphorylates
the inhibitory eukaryotic translation initiation factor 4E
binding proteins (4E-BP1 and 2) to detach them fromeukaryotic translation initiation factor 4E. The released
eukaryotic translation initiation factor 4E then aids the formation of the translation initiation complex at the 50 ends ofmRNA, thereby enhancing global translation (9). Besides theglobal translation stimulation, mTORC1 also boosts translation of mRNA bearing the 50 terminal oligopyrimidine tract(10). Moreover, mTORC1 phosphorylates ribosomal protein
p70 S6 kinase (S6K) that is required for the positive regulationof eukaryotic initiation factor 4A (eIF4A) and 4B (eIF4B) to
augment translation (11–14).
Cancer cells acquire the ability to induce protein synthesis,
growth, and proliferation by activating the translation
signaling pathways which facilitate the expression of proteinsrequired for cell survival and growth (15–17). In addition,translation induction can be caused by alteration of expression
and/or phosphorylation of diverse proteins involved in translation. For example, consecutive expression and phosphorylation of eukaryotic initiation factor 4F accelerated translation
initiation (15, 18–20). Reduced expression or increased
J. Biol. Chem. (2022) 298(7) 102084

1© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

mTORC1-independent translation control by MAT2A and SAM
phosphorylation of 4E-BPs promoted translation in pancreaticcancer (21). Overexpression of eukaryotic initiation factor 3
induced global protein synthesis and increased translation of
oncogenic transcripts in immortalized ﬁbroblastic cells (22).
Due to the contribution of mTOR to translation, cellular
growth, and proliferation, various drugs targeting the mTOR
pathway are being developed for cancer therapy (23–25).
However, one of the major problems of mTOR inhibition forcancer therapy is that upon inhibition of a protein in themTOR signaling, cancer cells develop a resistance to thechemotherapy by activating compensatory proteins by various
feedback mechanisms (26). For example, upon inhibition ofmTORC1, the AKT and mitogen-activated protein kinase
pathways are activated by feedback mechanisms to promote
cell survival (27–31). Therefore, it is of utmost importance toexplore new mTOR-independent translation regulatory
mechanisms to propose novel targets for cancer therapy.
The one-carbon metabolic cycle of methionine is intimately
connected with the mTOR signaling (32). It is known that themTOR signaling is responsive to S-adenosylmethionine(SAM), one of the one-carbon cycle metabolites, owing to the
SAMTOR protein acting as the SAM-sensor (33). SAM is
produced from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, three isozymes of MAT
are known (34, 35). MAT1 and MAT3 are found in liver,whereas MAT2 is expressed in all tissues except for adult liver(36–38). MAT2 consists of two subunits: MAT2A and
MAT2B, which form a complex with a molecular ratio of 2:1.
MAT2A is the catalytic subunit, whereas MAT2B has beensuggested to regulate the catalytic activity (39) or stability (40)of MAT2A. MAT2A is expressed in proliferating fetal hepatocytes and is replaced by MAT1 in adult quiescent hepatocytes (41). Upon partial hepatectomy, MAT2A is upregulated
during regeneration (42). Also, the expression of MAT2A is
known to be high in various cancers including those of liver(43, 44), uterine cervix (45), colon (46), and breast (47). Theseobservations suggest that MAT2A is required for cell growthand proliferation. Recently, MYC has been shown to induce
MAT2A expression (48), which explains how MAT2A is
upregulated in proliferative tissue and particularly in cancer.
These pieces of evidence suggest that SAM synthesis by
MAT2A is connected to cancer cell growth and proliferation.
However, whether the connection is mediated exclusively by
the mTOR pathway remains unknown.
SAM is the essential methyl group donor in biologicalmethylation of various biomolecules. Methylation in DNA,
RNA, and histone proteins is well-studied due to its criticalroles in gene expression control. Besides histones, nonhistone proteins such as ribosomal proteins and translation
factors are also methylated (49). Although the biologicalsigniﬁcance of methylation in ribosomal proteins is yet to beinterrogated, methylation of several translation factors is
known to regulate translation. For example, depletion of themethyltransferase for lysine 165 of eEF1A (eEF1A165K)
induced translation of proteins related to ribosome biogenesis
and chromatin. In contrast, it reduced translation of proteins

2 J. Biol. Chem. (2022) 298(7) 102084

important for unfolded protein response (50). Methylation oflysine 55 of eEF1A (eEF1A55K) promoted global translation
in a human lung cancer cell line (51). As some of the protein
components of translation machinery are methylated, we
hypothesized that MAT2 and SAM are coupled with protein
synthesis in mammalian cells by affecting their methylation.
In this study, we discovered that MAT2A promotes protein
synthesis in an mTORC1-independent manner. Polysome
proﬁling and RNA sequencing revealed that MAT2A isrequired for the formation of active polysome and maintenance of translation efﬁciencies of mRNAs. Moreover, wefound that MAT2A interacts with ribosome biogenesis factors and contributes to processing of the 18S rRNA and dynamic methylation of translation factors.

Results
MAT2A is essential for global protein synthesis in the
mammalian cells
To reveal the involvement of MAT2A in protein synthesis,
we examined the global protein synthesis rate upon Mat2aknockdown in the mouse hepatocellular carcinoma cell line
(Hepa1) by using surface sensing of translation (SUnSET) (52).
Elongating peptides in cultured cells were labeled for 30 min
with the aminoglycoside antibiotic puromycin. Translation
elongation is inhibited when translating ribosome incorporates
puromycin into nascent proteins. The resulting short peptides
labeled with puromycin can be detected by Western blotting
using an antipuromycin antibody, which enables examinationof the translation rate. We found that the global translation
was reduced in Hepa1 cells in which MAT2A was depleted bysiRNA (Figs. 1A and S1A). Treatment of cells with the translation inhibitor cycloheximide (CHX) greatly diminished thepuromycin-labeled proteins, indicating that puromycin speciﬁcally labeled the nascent peptides (Fig. 1A: Lanes 5 and 6).
In line with knockdown of Mat2a, chemical inhibition of
MAT2A by cycloleucine (cLEU), which is a substratecompetitive inhibitor of MAT2 (53, 54), signiﬁcantly reduced
protein synthesis in HeLa cells (Fig. 1B). Note that the
reduction in protein synthesis occurred within 1 h after the
MAT2A inhibition (Fig. S1B). Next, we examined the effect of
MAT2A overexpression on protein synthesis. We established
HeLa cells stably expressing FLAG-Bio-tagged MAT2A (FBMAT2A) (Fig. S1C). The FB-MAT2A protein was considered
as functional as the native MAT2A protein since immunoﬂuorescence microscopy using an anti-FLAG antibody
conﬁrmed that the FB-MAT2A was localized in nucleus as
expected (Fig. S1D), and streptavidin afﬁnity puriﬁcation of
FB-MAT2A showed its interaction with the endogenous
MAT2B protein (Fig. S1E). By single-cell colony isolation ofthese cells, a high FB-MAT2A-expressing clone was selected
for the following experiment. The FB-MAT2A-expressing cells
cultured under a normal condition showed higher protein
synthesis compared to the control cells established using theempty vector (FB-EV), indicating that MAT2A promoted
protein synthesis (Fig. 1C).

-

-

- -

+ +

(kDa)

(IB)
Puromycin labeled peptides

250
150

my
pa

Serum starvation
FB-MAT2A - + - + - + - + - +FB-EV + - + - + - + - + -

100
75

37

25
20
50

37
75

60
45
35

25

20
15

50

MAT2A
L: 1 2cLEU (mM)

3

4

5

6

0 10 20 30

37

140
100
75

(IB)
Puromycin
labeled peptides

75

(IB)
Phos-S6K(T389)

60
45
75

(kDa)

60
45

S6K

60
45
60

FB-MAT2A
Endo-MAT2A

45
45

35
25

GAPDH

35

45
35

L: 1

2

3

4

1.50

p = 0.001
p = 0.001
p = 0.001

1.25
1.00
0.50
0.25

0.00
cLEU (mM)

0Intensity(Puromycin labeled peptides/GAPDH)

L: 1

(IB)
β-ACTIN

Intensity(Puromycin labeled
peptides/β-ACTIN)

(kDa)
180
100
75

(IB)
Puromycin labeled peptides

50

(IB)
β-ACTIN

B

Serum stimulation
1 hr 3 hr

Ra

-c
si
CHX

cin

C

on

A

tro
l
si
-M
AT
2
si
-c A
on
si tr
-M o
AT l
2A

mTORC1-independent translation control by MAT2A and SAM4

2 3 4 5 6 7

8 9 10p = 0.040
p = 0.019

3

p = 0.045

p = 0.101
p = 0.023

2
1
0
L:

1

2

3

4

5

6

7

8

9 10

10 20 30

Figure 1. MAT2A is obligatory for global protein synthesis in mammalian cells. A, SUnSET method revealed a reduction in global translation upon
MAT2A knockdown in Hepa1 cells (lanes 3 and 4) compared to control (lanes 1 and 2). Cycloheximide treatment (CHX, at 50 μg/ml for 4 h) ensured thespeciﬁcity of the anti-puromycin antibody (lanes 5 and 6). B, MAT2A inhibition by cLEU reduced protein synthesis. HeLa cells were treated with cLEU at theindicated concentrations for 6 h (top panel). The intensity of puromycin-labeled peptides on the blot was quantiﬁed and was normalized to that of β-ACTIN(bottom panel). Mean values ± SD are shown (n = 4 biological replicates of the experiment). C, SUnSET assay was performed with HeLa cells stably
expressing FB-MAT2A in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of rapamycin (400 nM for 1 h). Cells were cultured in serum-free media for
24 h (serum starvation, lanes 5 and 6) and then were recultured in 10% FBS-containing media for the indicated periods (serum stimulation, lanes 7–10) (toppanel). The intensity of puromycin-labeled peptides on the blot was quantiﬁed and was normalized to that of GAPDH (bottom panel). Mean values ± SD are
shown (n = 3 biological replicates of the experiment). cLEU, cycloleucine.

MAT2A-regulated protein synthesis is independent of mTORC1
activity
To investigate whether the MAT2A-promoted protein
synthesis involved mTOR, we cultured these cells underserum starvation for 24 h and then restimulated the cells withserum. Serum starvation did not affect protein synthesis inthese cells. Restimulation of the starved cells with serum for
3 h increased protein synthesis in the FB-MAT2A-expressing

J. Biol. Chem. (2022) 298(7) 102084

3

mTORC1-independent translation control by MAT2A and SAM
cells but not in the control cells (Fig. 1C, lanes 5–10).
Importantly, the effect of FB-MAT2A was not diminished by
the treatment with rapamycin. These results indicate that
MAT2A does not rely on the mTORC1 activity to promote
protein synthesis. mTORC1 is activated by various stimuli
including growth factors and nutrients such as amino acids,
exerting effects on translation machinery (Fig. S2). To further
investigate whether MAT2A affects the mTOR signaling
pathway, we examined the activation status of mTORC1 upon
MAT2A perturbations. S6K is the downstream target ofmTORC1. Since the amount of phosphorylated S6K (PhosS6K) directly reﬂects the activity of the mTORC1 signaling(55), we examined the amount of Phos-S6K by Western
blotting. The FB-MAT2A-expressing cells did not show any
appreciable alteration in Phos-S6K compared to the control
cells under the normal conditions or when the cells werestarved and were restimulated with serum (Fig. 1C). PhosS6K was unchanged upon MAT2A inhibition by cLEU in
both HeLa (Fig. 2A) and Hepa1 (Fig. 2B) cells, whereas it was
reduced in response to rapamycin. These results suggest thatthe effect of MAT2A on protein synthesis is not contingent
on the activity of mTORC1.
MAT2A and SAM are essential for maintaining active
translation in cells
To elucidate the characteristics of the MAT2A-regulated
protein synthesis, we carried out a polysome proﬁling of
HeLa cells upon MAT2A inhibition by cLEU. Heavy polysome(also called active polysome) formation was impaired upon
MAT2A inhibition with a concomitant increase of monosome(80S) and its subunits (40S and 60S) (Fig. 3A). These characteristics of polysome proﬁle suggest that translation initiation
to form monosomes was hampered, and only a smaller fraction

Hepa1 cells

cLEU (mM)
0

10

20

30

mTOR
Phos-mTOR(S2448)Phos-S6K(T389)

kDa
300
250
180
300
250
180
100
75

Phos-S6K(T389)

60

S6K

(IB)

Rapamycin

Rapamycin

DMSO

To understand the mechanism of the regulation of protein
synthesis by MAT2A, we aimed to elucidate the MAT2Ainteracting protein network. We puriﬁed FB-MAT2A fromHeLa cells stably expressing FB-MAT2A by afﬁnity puriﬁcation with streptavidin. The puriﬁed proteins were then separated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE), were in-gel trypsin-digested, andB

HeLa cells(IB)

MAT2A interacts with proteins involved in ribosome
biogenesis and translation

DMSO

A

of successfully formed monosomes proceeded to active polysome formation.
To investigate the MAT2A-regulated translational control,
we isolated translating mRNA from the polysome fractions
and performed RNA sequencing. We found that after MAT2A
inhibition, 507 transcripts were upregulated, and 193 were
downregulated in the polysome occupancy (Fig. 3B and
Table S1). The translation efﬁciency (TE) of each mRNA wascalculated by a ratio of reads number between polysome
fraction and total RNA, which showed that the number oftranscripts with decreased TE (135) by MAT2A inhibition was
greater than that of transcripts with increased TE (120)(Fig. 3C and Table S1). Next, we performed RNA-sequencingof the cells treated with or without cLEU. A total of 1147 and328 transcripts, respectively, were upregulated and downregulated signiﬁcantly (q < 0.01) in total RNA fractions after
MAT2A inhibition (Fig. 3D and Table S1). Gene ontology(GO) analysis revealed that the expression of genes involved inribosome biogenesis was reduced in the cLEU-treated cells(Fig. 3E). These results strongly suggested the existence of atranslation regulation mechanism involving MAT2A and
SAM, which concerns the expression of a speciﬁc subset ofmRNA through the altered formation of polysome.

cLEU (mM)
0
30

kDa
75
60
45
75
60

45
100
45
45

75

S6K

60

GAPDH

35

45
45

GAPDH

35

25

L:

1

2

3

4

5

6

25

L:

1

2

3

4

5

6

Figure 2. MAT2A-regulated protein synthesis is independent of mTORC1. A and B, Western blotting for the mTOR pathway proteins after treating cells
at different concentrations of cLEU for 6 h using HeLa (A) and Hepa1 (B) cells. In lane 2, cells were treated with 400 nM rapamycin for 1 h. cLEU, cycloleucine;mTORC1, mechanistic target of rapamycin complex 1.

4 J. Biol. Chem. (2022) 298(7) 102084

mTORC1-independent translation control by MAT2A and SAM
MonosomeA

Control
60S

Absorbance (260 nM)

0.9

0.6

cLEU

40S

Light
polysome

Heavypolysome

0.3

0

10

0

20

30

40

50

60

70

80

90

Distance (mm) on 10-50% sucrose gradient
-0.3

B

C

cLEU treatment (q<0.01)
Up transcripts: 507

2
1
0

-1
-2
Down transcripts: 193

-3
1x100

1x102

1x104

mRNA changes in total fraction[log2]

50-5> 2-folds downtranscripts: 135

5

10

15

20

E
GO Term:
Biological Process p.adjust

Up transcripts: 1147

2e−08
4e−08
6e−08
8e−08

Ribosome biogenesis

1

25

Mean mRNA expression (log2)2ncRNA processing

0

-1
-2

> 2-folds up
transcripts: 120

0cLEU treatment (q<0.01)
3

10

- 10

1x106

Mean mRNA expression

D

cLEU treatment

Translation efficiency change(log2) [log2]mRNA changes in polysome
fraction [log2]

3

5

10 15 20
Gene countDown
transcripts: 328

-3
1x100
1x102
1x104
1x106
Mean mRNA expression

Figure 3. MAT2A and SAM are essential for maintaining active translation in cells. A, representative merge of polysome curves derived from control(blue) and 6-h cLEU–treated (magenta) cells. The experiment was repeated three times, which resulted in polysome curves with similar morphology. B, RNAsequencing was performed using mRNA extracted from the polysome fractions in (A). Differential expression analysis was performed for the polysomebound transcripts exhibiting ten or more read counts. Differences between the control and treated group were considered signiﬁcant when q value
was smaller than 0.01. C, translation efﬁciency (TE) of each transcript was calculated by a ratio of reads number between the polysome fraction in (B) andtotal RNA. Transcripts exhibiting 2-fold (Log2) or more increments are colored red, and those exhibiting 2-fold (Log2) or more decrements are shown in blue.
D, RNA sequencing was performed using the total cytoplasmic mRNA from the control cells and cells treated with cLEU. Differential expression analysis was
performed for the genes exhibiting ten or more read counts. The genes whose expression were signiﬁcantly altered upon the treatment are indicated indark gray. Differences between the control and treated group were considered signiﬁcant when q value was smaller than 0.01. E, Gene ontology analysis of
the transcriptionally downregulated genes shown in (D). Two signiﬁcantly enriched terms (p.adjust. < 0.01) are presented in a horizontal bar plot. p.adjust:adjusted p-value. n = 2 in all the analyses. cLEU, cycloleucine.

were analyzed by liquid chromatography with tandem mass
spectrometry (LC-MS/MS) (Fig. 4A). Coomassie Brilliant Blue
(CBB) staining (Fig. 4B) and Western blotting (Fig. 4C) of the

puriﬁed protein showed that the puriﬁcation successfully
enriched proteins interacting with FB-MAT2A. We found that81 proteins were speciﬁcally copuriﬁed with FB-MAT2A
J. Biol. Chem. (2022) 298(7) 102084

5

mTORC1-independent translation control by MAT2A and SAM

kDaC
Input Purified
FB-EV
FB-MAT2A
FB-EV
FB-MAT2A

Stable expression of
FB-MAT2A in HeLa cells

FB
-E
V
FB
-M
AT
2A

B

A

250

Straptavidine purification

150
100
75

SDS-PAGE

kDa
75

MAT2A
MAT2B

50
37

In-gel trypsin digestionand peptides extraction

(IB)
MAT2A

50
37

25
15

LC-MS/MS analysis

D

GO Term: Biological Process
p.adjust

Ribonucleoprotein complex biogenesisrRNA processing
rRNA metabolic process
Regulation of mRNA processing
Cytokinesis
Positive regulation of phosphoprotein phosphatase activity
Regulation of cyclic−nucleotide phosphodiesterase activity

1e-04
2e-04
3e-04
4e-04
5e-04

(Protein count) 0

5

10

15

20

E
RPF1

RRP1
SRSF10

IMP4

SRSF12HNRNPDLE

RBMX

PPP1R12A

SERF2

ACIN1
MYH11

SPTBN1
CALM2

WDR46

TMA7

RBM3
ZNF326EPPK1

MAT2A interacting proteome related
to ribosome biogeneis or function

PHF14

GNB1

FB-MAT2A
His-EV
His-IMP4
(IB)
MAT2A

+
+
-

PESE1
LIMCH1

CBX5
SONE

SAFB

25

RPL30
CTTN

0

DSG2
PHF14
SNRPAE

NUMA1
MKI67

LMNB2
IMMT

Inputs

SEC61A1
MYL3
MYH14

DYNLL1
MYL1

DDX41

MAT2A

LIMA1

MYO1B

F

CLTC

BST2

EIF3I
BMS1

FAM32A

CALM1

STOM
SNU13

Protein scorefold change
LMO7(over control)
50
CORO1B

SAFB2

MAT2B

CALM3

RRS1

NOP56

SPTAN1
CFL1

LMNB1

NHP2

GNAI3
MASTLEEDF1

RBMXL1PNN
DDX54

snoRNP
complex

RBX1

RBMXL2

PDCD11

NOP58

SAP18

GNG12
PRC1

PSIP1

EBNA1BP2

RBM34
RBM14

MYBBP1ARRP12CENPVE

Purified

+ +
- +
+ -

+
+

(kDa)
60

45
45

His-IMP4
35

L:

1

2

3

4

Figure 4. MAT2A interacts with proteins involved in ribosome biogenesis and translation. A, schematic representation of the workﬂow for ectopic
expression and puriﬁcation of FB-MAT2A for LC-MS/MS analysis. B, FB-MAT2A and its interacting proteins were visualized by Coomassie Brilliant Blue (CBB)
staining following SDS-PAGE. C, conﬁrmation of puriﬁcation of FB-MAT2A by Western blot using a monoclonal MAT2A antibody. D, Gene ontology (GO)analysis for the FB-MAT2A interacting proteins. Top seven enriched terms are presented in a horizontal bar blot for GO-term Biological Process. The color
grades indicate the adjusted p-value for each enriched term. E, FB-MAT2A interacting proteins are presented as a network. The color grade indicates the foldchange of protein score over control puriﬁcation. The proteins in the left rectangular area are related to ribosome biogenesis or function. Proteins inside thedashed black circle are the components of the snoRNP complex. The IMP4 protein is indicated by a black arrow. F, copuriﬁcation of His-IMP4 with FB-MAT2A
by streptavidin afﬁnity puriﬁcation. The Western blotting was done using monoclonal mouse anti-MAT2A and polyclonal rabbit anti-His tag antibodies.

(Table S2). In the result of GO analysis, the top enriched terms
were related to ribosome biogenesis and/or functions such as
ribonucleoprotein complex biogenesis, rRNA processing,rRNA metabolic process, preribosome, small nucleolar ribonucleoprotein complex, and snoRNA binding (Figs. 4D and S3,

6 J. Biol. Chem. (2022) 298(7) 102084

A and B), suggesting an involvement of MAT2A in ribosome
biogenesis and/or ribosome function. Notably, the core protein
components of the snoRNP complex (NOP56, NOP58 and
SNU13), except for FBL (Fibrillarin), were found in the puriﬁed proteins (Fig. 4E). The snoRNP complex plays an

mTORC1-independent translation control by MAT2A and SAM
important role in ribosome biogenesis by 20 -O-methylation ofrRNA, and this methylation also enhances the translation
capability of ribosome (56). These results strongly support theimperativeness of MAT2A in the ribosome function.
MAT2A is required for rRNA processing
Mammalian ribosomal RNA is transcribed as a single largetranscript called the 47S pre-rRNA. This pre-rRNA bears
external and internal transcribed spacers (ITSs), which must
be removed by the complex process of endonucleolytic andexonucleolytic cleavages at speciﬁc sites to produce the mature
18S, 5.8S, and 28S rRNAs (57) (Fig. S4A). Among the MAT2Ainteracting ribosome biogenesis factors, we further conﬁrmed
the interaction of IMP4 with MAT2A by copuriﬁcation ofpoly-histidine-tagged IMP4 (His-IMP4) with FB-MAT2A(Fig. 4F). IMP4 is a component of the small subunit processome. The small subunit processome mediates 18S rRNA
maturation by cleavage at A0, A1, and A2 sites (58). To
elucidate whether MAT2A is required for the pre-rRNA
transcription, we quantiﬁed the pre-rRNA content in Mat2aknocked-down Hepa1 cells by qPCR using a primer pair targeting the 50 external transcribed spacer sequence (59). We
observed that Mat2a knockdown did not alter the rRNA
transcription in Hepa1 cells (Fig. S4B), suggesting that
MAT2A is dispensable for rRNA transcription. MAT2A
interacted with the snoRNP complex which plays a critical role
in rRNA methylation and maturation (60–62) (Fig. 4E). We
also observed the interaction of MAT2A with IMP4, one of the
18S rRNA cleavage factors. These observations led to a hypothesis that MAT2A might be involved in rRNA maturation.
Indeed, Mat2a knockdown decreased the 18S/28S rRNA ratiowith reduced production of the 18S rRNA (Fig. 5, A and B).
Chemical inhibition of MAT2A by cLEU also reduced the 18S/
28S ratio in a concentration-dependent manner (Figs. 5C andS4C). Alterations in rRNA processing were further investigated by measuring the major pre-rRNA stages by Northernblot using ITS1 and ITS2. The reduced production 18S rRNA
upon the cLEU treatment was supported by the result that aweakened signal of 18S-E, the immediate precursor of themature 18S rRNA, was detected using the ITS1 probe (Fig. 5D,
Panel 1). Interestingly, we observed a prominent accumulationof 30S and 32S pre-rRNAs, the precursors of the mature 18S
rRNA (Fig. 5D, Panels 1 and 2). The decreased 18S rRNA wasalso observed (Fig. 5D, Panel 3). Considering that rRNA
transcription was not affected by MAT2A knockdown, thereduced production of the 18S rRNA could be a result of
disturbed 18S rRNA processing. These observations suggestthat MAT2A is involved in 18S rRNA cleavage and processing.
Insufﬁcient 18S rRNA maturation may have partiallycontributed to the reduction of protein synthesis upon
MAT2A depletion or inhibition.
MAT2A contributes to dynamic methylation of proteins
involved in translation
Many proteins involved in translation are methylated in
E. coli, yeast, and mammals (49, 51, 63–66). Although some ofthese methylations appeared important for translation (50,51, 67), the biological signiﬁcance of the majority is unclear.
Therefore, we hypothesized that a reduction of methylation ofproteins involved in translation might cause the protein
synthesis reduction upon MAT2A inhibition. To compare
methylation of proteins upon MAT2A inhibition, we
employed a variant approach of stable isotope labeling byamino acid in cell culture (SILAC) named heavy methyl
SILAC (68) followed by identiﬁcation and quantiﬁcation of
methylated peptides using LC-MS/MS. HeLa cells werelabeled with either light or heavy methionine for six generations. The heavy-labeled cells were treated with 30 mM cLEU
for 6 h and the light and heavy-labeled cells were then mixed
at a ratio of 1:1. Then, ribosomes were puriﬁed by two
methods for the maximum coverage (Fig. 6A): by immunoprecipitation (IP) (method 1) or ultracentrifugation in sucrose
cushion (method 2). In method 1, cells were transfected withthe 3× FLAG-tagged RPL23A protein (FL-RPL23A) expression vector and then ribosome containing the FL-RPL23A
protein was puriﬁed by ani-FLAG IP. For comparison, amock puriﬁcation was also performed in parallel using cells
transfected with the empty vector (Fig. S5, A and B). In
method 2, ribosome was pelleted by passing the cleared lysate
through 1 M sucrose cushion in ultracentrifugation (Fig. S5,
C and D). We noticed that many ribosomal proteins were
puriﬁed in both methods (Table S3). By combining the dataobtained from the two methods, we identiﬁed 28 methylated
peptides (Table S3; Sheet 3). After calculating the heavy/lightratio from the relative abundance of methylated peptides,
the values were normalized to those of unmethylated
methionine-containing peptides derived from the same protein to exclude the possible variability due to the difference incell number or protein amount between light-labeled andheavy-labeled cells. When no unmethylated methioninecontaining peptide was identiﬁed in the same protein, thenormalization was done using RPL23A (method 1) or
PABPC1 (method 2) as the standards (see Supplementary
Method for details) (Table S3). By taking average abundance values of peptides puriﬁed twice, we ﬁnally identiﬁed
16 unique peptides (Table S3; Sheet 5). Among the uniquepeptides, 12 peptides were derived from proteins involved intranslation, and ﬁve of them showed a reduction by 60% ormore in methylation upon MAT2A inhibition by the cLEU
treatment for 6 h (Fig. 6B and Table S3). The relative abundance of methionine-containing peptides used for normalization showed a linear correlation in both methods (Fig. 6, C
and D), indicating that the same amount of proteins were
puriﬁed from the control and treatment samples. These results suggest that MAT2A contribute to dynamic methylationof a speciﬁc set of proteins for translation.
The highest methylation reduction was observed in
K55me2 of eEF1A1 and/or eEF1A2. The chromatogram andmass spectrum of eEF1A1/2K55me2 light and heavy peptides
are presented in Figure 7A. We further validated the effect of
MAT2A on eEF1A1/2K55me2 by Western blot using a speciﬁc antibody for this methylation. For convenience oftransfection, we selected HEK293T cells for this experiment.
J. Biol. Chem. (2022) 298(7) 102084

7

mTORC1-independent translation control by MAT2A and SAM

B

18S rRNA

Marker RNA

28S rRNA

18S rRNA

20 25 30 35 40 45 50 55 60
Time (S)28S rRNA

si-MAT2A

si-Control

1000
900
800
700
600
500
400
300
200
100
0

Marker RNA

Fluorescence intensity

A

20 25 30 35 40 45 50 55 60
Time (S)C0.8

p = 0.0018
rRNA ratio (18S/28S)

0.6
0.4
0.2
0.0

0.4
0.2

AT
2

0

10

-M

30S

ControlcLEU

ControlcLEU

ControlcLEU

47S/45S
41S

20

30

cLEU (mM)

si

-C

on

tro

l

A

0.0

si

D

0.6

ControlcLEUrRNA ratio (18S/28S)

0.8

47S/45S
41S
32S
28S

26S
21S
18S-E

18S

180
160
140
120
100
80
60
40
20
0

Intensity(Control vs cLEU treatment)

47S/45S 30S 32S 18S-E

12S
Control

47S/
45S

30S

32S

18S-E

100

100

100

100

Average

Probe: ITS1
Panel: 1

ITS2
Panel: 2

18S
Panel: 3cLEU 123.6 149.4 112.5 79.9

28S
Panel: 4SE

Control

0

0

0

0

cLEU 12.5

5.8

11.1

9.6

Figure 5. MAT2A is required for rRNA processing. A and B, the mature 18S and 28S rRNA were quantiﬁed after knockdown of MAT2A in Hepa1 cells. The
representative electropherograms from triplicated samples are presented in (A). A bar plot showing a signiﬁcant reduction of the 18S/28S rRNA ratio ispresented in (B). Mean ± SD are shown (n = 3 replicates of samples). C, the decreased 18S/28S rRNA ratio was also observed in HeLa cells after chemical
inhibition of MAT2A by cLEU. D, panel 1 and 2: Northern blotting showing different pre-rRNA detected by the ITS1 and ITS2 probes. Panel 3 and 4: The
mature 18S and 28S rRNAs are detected by speciﬁc probes. The bar plot on the right side and the table below indicate the relative quantity of each pre-rRNA
that was changed upon cLEU treatment. cLEU, cycloleucine; IP, immunoprecipitation; ITS, internal transcribed spacers.

Because of its ubiquitous expression (69), the principal formof eEF1A in HEK293T cells is eEF1A1. Therefore, we examined the methylation status of eEF1A1K55me2 in HEK293T
cells upon MAT2A transient overexpression. First, we validated the speciﬁc reactivity of commercially available
DiMethyl-eEF1A-K55 antibody (anti-eEF1AK55me2) byoverexpressing either wildtype FB-eEF1A1 or mutant FBeEF1A1K55R (lysine was replaced by arginine) in HEK293T

8 J. Biol. Chem. (2022) 298(7) 102084

cells followed by IP with the FLAG antibody and immunoblotting with the anti-eEF1AK55me2 antibody. We observedthat the anti-eEF1AK55me2 antibody detected wildtype FBeEF1A1 both in the input (Fig. 7B, lane 2) and IP (Fig. 7B,
lane 5) samples but failed to detect the corresponding signalof FB-eEF1A1K55R (Fig. 7B, Lanes 3 and 6, respectively),
corroborating that this antibody speciﬁcally detects
eEF1A1K55me2. Next, we co-expressed either wildtype FB-

mTORC1-independent translation control by MAT2A and SAM

A

Light

Method 1

Heavy[13CD3]Methionine[CH3]Methionine

MATs

MATs

18 hr

MTs

[CH3]proteins[13CD3]proteinsB

LC-MS/MS

6 hr

Transfection

Mix

Ribosome
IP

Method 2[CH3]-SAM [13CD3]-SAM
MTs+ cLEU+ cLEU

LC-MS/MS

6 hr
Mix Ribosome
pelleting

Methylated peptides

Proteins(Types of modification)

GSFKYAWVLDKLK eEF1A1/2 (K55me2)
SGVGNIFVKNLDK PABPC3 (K104me1)(K361me1)
IVGTKPLYVALAQR PABPC1L(K361me1)
IVGTKPLYVALAQRK PABPC1L(K525me3)
VAVEAKNPADLPKLVEGLKReEF2
VANTSTQTMGPRPAAAAAAATPAVRKQSGYGGQTKPIFRKKQSGYGGQTKPIFRQSGYGGQTKPIFRGLDVEDVKFVINYDYPNSSEDYVHRIGR
VMSTQRVANTSTQTMGPRPAAAAAAATPAVRAAVAAAAPRAVAPYKYASSVR

0.0

D

C
28

(R493me2)(K89me1)(K89me1)(K89me1)(R505me1)(R493me2)(R534me2)

PABPC1
RPL36A
RPL36A
RPL36ADDX17PABPC1PABPC40.2 0.4 0.6 0.8 1.0
Area ratio (Heavy/ Light)

20.0

PABPC1PABPC4
eEF2

Light area (log2)

Light area (log2)

PABPC126

DDX17

RPL23A24

1.2

RPL23APABPC1

eEF2

27.5

PABPC1PABPC1PABPC3

25.0

eEF2

PABPC3PABPC422.5

22

eEF1A1/2

y = 2.41 + 0.922 x R²adj = 0.94
22

24
26
Heavy area (log2)

28

17.5
17.5

y = 1.35 + 0.948 x R²adj = 0.99
20.0
22.5
25.0
Heavy area (log2)

27.5

Figure 6. MAT2A contributes to dynamic methylation of proteins involved in translation. A, schematic diagram of heavy methyl-SILAC and puriﬁcationof ribosomes by IP (method 1) or by pelleting in ultracentrifugation (method 2). B, the identiﬁed methylated peptides derived from proteins involved intranslation are shown. The methylated residues are shown in magenta. The horizontal bar plot indicates the heavy/light ratio of each peptide. A ratio smaller
than 1 indicates reduced methylation in the peptide by cLEU treatment. The bars of peptides whose methylation was reduced by more than 60%(ratio < 0.6) are presented in blue. The type of modiﬁcation is labeled at the tip of each bar. Kme1, monomethylated lysine; Kme2, dimethylated lysine;Kme3, trimethylated lysine; Rme1, monomethylated arginine; Rme2, dimethylated arginine. C and D, correlation analysis between the relative abundance oflight and heavy unmethylated methionine-containing peptides used for normalization in methods 1 and 2 are shown in C and D, respectively (two peptides
from PABPC1, two from RPL23A, and one from DDX17 in C and one peptide from eEF1A1/2, three from eEF2, three from PABPC1, two from PABPC3, and one
from PABPC4 in D). The gray zone indicates the 95% conﬁdence level interval predicted from a linear model (“lm”) in R. cLEU, cycloleucine; IP, immunoprecipitation; MAT, methionine adenosyltransferase; SILAC, stable isotope labeling by amino acid in cell culture.

eEF1A1 or mutant FB-eEF1A1K55R with MAT2A in
HEK293T cells. As expected, we observed that the expressionof MAT2A increased eEF1A1K55me2 (Fig. 7C, lane 5). Thisobservation was consistent with the reduction in
eEF1AK55me2 upon MAT2 inhibition observed in the heavy
methyl-SILAC experiments.

Discussion
In this study, we examined the role of MAT2A in protein
synthesis in mammalian cells. We revealed that MAT2A andSAM are essential to maintain global protein synthesis independently of the mTORC1 signaling. In the polysome proﬁling
experiment, we observed that inhibition of the MAT2A activity

J. Biol. Chem. (2022) 298(7) 102084

9

mTORC1-independent translation control by MAT2A and SAM

Figure 7. MAT2A induces methylation of eEF1A1 at lysine 55. A, the chromatogram (upper panels) and mass spectrum (lower panels) of light and heavy
eEF1A1/2K55me2 peptides are presented. The retention time (RT), area (AA), and m/z of this peptide are presented in blue (light peptide) and magenta(heavy peptide). B, the speciﬁc reactivity of the anti-eEF1AK55me2 antibody was determined. The eEF1A1 WT and the methylation-mutant protein weretransiently expressed for 24 h in HEK293T cells, and IP and immunoblotting were done using the indicated antibodies. The magenta arrows indicate ectopic
FB-eEF1A1. C, the eEF1A1 WT and methylation-mutant proteins were co-expressed with MAT2A in HEK293T cells for 24 h, and immunoblotting with theindicated antibodies was done. The magenta arrows indicate ectopic FB-eEF1A1. This experiment was repeated two times, which resulted in the sameobservation.

impaired the formation of active polysome and altered TE of asubset of mRNA. To understand the mechanistic aspects, westudied the MAT2A interactomes by mass spectrometry. We
observed that MAT2A interacts with many rRNA processing
and ribosome biogenesis factors. We revealed that the 18S
rRNA maturation was severely affected by MAT2A depletion
and inhibition. By quantitative mass spectrometry, we showed adrastic reduction of methylation of translation factors including

10 J. Biol. Chem. (2022) 298(7) 102084

eEF1A1 in MAT2A-inhibited cells. To sum up, MAT2A andSAM promote translation in mammalian cells by (i) facilitatingrRNA processing, (ii) promoting active polysome formation,and (iii) regulating methylation of translation factors (Fig. 8).
Importantly, the effect of MAT2A and SAM on translation is
independent of the mTORC1 signaling.
We observed a reduction in protein synthesis not only upon
MAT2A depletion but also by chemical inhibition of MAT2A

mTORC1-independent translation control by MAT2A and SAM
Mammalian Cell

Nucleus

Nucleolus

rDNA

Pol-I

Transcription
5´ ETS

18S

5.8S

ITS1

ITS2

28S

Pre-rRNA

3´ ETS

Processing &Modification
5.8S

18S

28S

Methionine
MAT2A
SAM

MaturerRNA

Assembly and export
Cytoplasm
Translation
factors

28S
5.8S 5S
pre-60S

18S
pre-40S

M

M

M
et
hy

la
tio
n

Translation initiation

M

?

M

M

SA
AT M
2A

M

?

60S

mRNA

40S
Polysome formation

Methionine
MAT2A
SAM
Methionine
MAT2A
SAM

Methionine

Figure 8. MAT2A contributes to translation by multipronged mechanisms: promotion of rRNA processing in the nucleolus, promotion oftranslation initiation and active polysome formation in the cytoplasm,and dynamic methylation of some translation factors. ITS, internaltranscribed spacers; SAM, S-adenosylmethionine.

activity, indicating that MAT2A relies on SAM for promoting
protein synthesis. It has been reported that SAM and its precursor methionine are necessary for proliferation of cancer
cells (70–72) and stem cells (73–75). Moreover, SAM scarcity
in cancer leads to cell cycle arrest in G1 and G2 phases where
cells synthesize proteins, lipids, and other biomolecules(76, 77). In addition, cell proliferation requires the synthesis ofa copious amount of protein. Therefore, our data suggest that
MAT2A and SAM contribute to cell proliferation by maintaining protein synthesis. On the other hand, several studies
have depicted that an excess of exogenous SAM supplementation can be toxic and suppresses proliferation of cell lines
from various cancers including uterine cervix (78), colon (79),colorectal (80), and breast (81) by arresting cell cycle in S
phase (80, 82). Therefore, an optimum amount of SAM isnecessary for protein synthesis and cell cycle progression.
In this study, we found that MAT2A regulates translation
independently of the mTORC1 activity. Contrary to our
ﬁndings, a previous study showed that the activity of mTORC1
is highly dependent on cellular SAM concentration. It wasreported that when SAM becomes low in HEK293T cells,
SAMTOR inhibits the mTORC1 activity by forming a stable
dimer with GATOR1. Under a sufﬁcient SAM condition, SAM
binds to SAMTOR to disrupt the SAMTOR-GATOR1 complex, thereby activating mTORC1 and translation (33). While
our ﬁndings are contradictory to this report, the difference
may be caused by the SAM depletion techniques employed.
They carried out methionine restriction for 2 h in theirexperiment. The activation of mTORC1 requires its localization on the lysosomal surface, where the activator RHEB

resides, and the localization is stimulated by an amino acidsignaling (28, 83). Therefore, the observation by Gu et al.
might be confounded by a compromised amino acid signalingrather than solely depending on SAM concentration. Further
studies are required to clarify these possibilities. It should be
noted that serum stimulation increased MAT2A protein(Fig. 1C). This observation may suggest that mTOR induces
the expression of MAT2A.
Paradoxically to the drastic protein synthesis reduction, TE
of only a small number of transcripts was decreased in
MAT2A-inhibited cells. This observation can be explained by
the fact that MAT2A is important for global translation rather
than that of speciﬁc mRNA. Therefore, a small reduction ofTE, which was not considered for calculation in Figure 3C, formany genes combinedly might lead to the huge protein synthesis reduction.
The MAT2A-interacting proteins included many rRNA andribosome biogenesis factors, particularly the components ofthe snoRNP complex, which mediates rRNA methylation(Fig. 4E). We conﬁrmed the interaction of MAT2A with IMP4.
In addition, the 18S rRNA processing was reduced upon
MAT2A depletion (Fig. 5, A and B) or inhibition (Fig. 5, C andD), whereas rRNA transcription was not affected (Fig. S4B).
The necessity of methylation for rRNA biogenesis (61, 84–86)
suggests that the 18S rRNA defects observed upon MAT2A
depletion or inhibition are explained by impaired methylation.
The interaction of MAT2A with the snoRNP complex may
expand the idea of the local SAM synthesis and consumption
model of chromatin methylation in which SAM is produced on
chromatin for local histone methylation (87, 88). MAT2A may
produce SAM for rRNA methylation along with its processing.
Further studies including rRNA methylation are necessary to
draw an absolute conclusion.
Posttranslational methylation is one of the most common
modiﬁcations found in a wide range of proteins. Given thattranslation is one of the critical steps for gene expression
control, methylation of ribosomal proteins and translation
factors might be crucial for the translational regulation ofgene expression. By quantitative mass spectrometry, we
revealed that methylation of some translation factors, such as
eEF1A1/2K55me2, PABPC3K104me1, PABPC1LK361me1,
and eEF2K525me3, were reduced by more than 60% upon
MAT2A inhibition (Fig. 6B). We must note that some ofthese identiﬁcations and quantiﬁcations may have been
disturbed by distinct unmethylated peptides derived fromhomologous proteins in which amino acid replacements alter
the number of the methyl group, such as the replacement of
Gly by Ala (see Table S3, sheet 3, column AE). We further
validate the effect of MAT2A on eEF1A1K55me2 by using a
speciﬁc antibody (Fig. 7, B and C); transient overexpression
MAT2A induced eEF1A1K55me2. These observations suggest that methylations of these proteins are dynamicallyaltered in response to intracellular SAM concentration.
It has been reported that methylations of some of these
proteins are necessary for translation. For example,
eEF1AK55me2 is required for protein synthesis and tumorigenesis in lung cancer (51) and cell proliferation in acute
J. Biol. Chem. (2022) 298(7) 102084

11

mTORC1-independent translation control by MAT2A and SAM
myeloid leukemia (89). It is also reported that eEF1AK55me2
has a more speciﬁc function in enhancing the translation ofcodons for tryptophan and alanine (90). Therefore, MAT2A
may at least partially rely on eEF1AK55me2 to stimulate
protein synthesis. eEF2K509me3 (the corresponding residue inhuman is K525) is essential for maintaining translation framein yeast (67). Although there is no report of PABPC1L
methylation as far as we know, upregulation of PABPC1L is
associated with proliferation and migration of prostrate andcolorectal cancers (91, 92). Therefore, it would be promising toinvestigate the importance of PABPC1LK361me1 in translation. PABPC3 and PABPC4 are known to be methylated atseveral lysine and arginine residues (93, 94). However, thebiological signiﬁcance of these methylations is still unknown.
Since PABPCs have translation-stimulating capabilities(95–97), the biological signiﬁcance of their methylations would
be promising to be explored in future.
In this study, we focused on methylation of translationrelated proteins. tRNA is also highly modiﬁed by methylation at different positions, and some of them have alreadybeen proven important for various steps of translation(98–100). Therefore, in future, it will be propitious to investigate whether and how MAT2A and SAM contribute totRNA-modiﬁcation.
Due to the complex nature of the mTOR signaling, thecancer chemotherapy targeting the mTOR signaling pathways
for translation control is often compromised by different
mechanisms including mutations of inhibitor binding sites(101, 102), activation of costimulatory molecules (103),
induced expression of growth factor receptors (104), andreduced expression of inhibitory molecules (105). Furthermore, the mTOR inhibitors compromise cancer therapy due totheir potent immunosuppressive properties (106). Under thesecircumstances, a therapy targeting MAT2A could be analternative to suppress the translation for cancer therapy.

Experimental procedures
Cell culture
Hepa1 and HeLa cells were cultured in Dulbecco’s Modiﬁed
Eagle’s Medium (DMEM) containing 4.5 g/l glucose (D5796,
DMEM-high glucose, Sigma-Aldrich) supplemented with 10%
fetal bovine serum (FBS) (172012, Sigma-Aldrich) and combination of 100 U/ml penicillin and 100 μg/ml streptomycin(15140-122, penicillin-streptomycin, Thermo Fisher Scientiﬁc). In polysome analysis, HeLa cells were cultured in DMEM
media containing 4.5 g/l glucose (08458-16, Nacalai Tesque)supplemented with 10% FBS (26140079, Thermo Fisher Scientiﬁc). HEK293T cells were cultured in DMEM containing
1.0 g/l glucose (D6046, Sigma Aldrich) supplemented with 10%
FBS (172012, Sigma Aldrich).
Plasmids
The pEF1α-FLAG-Biotag empty vector (FB-EV), pEF1αFLAG-Biotag-MAT2A (FB-MAT2A), and pEF1α-FLAG-Biotag-BirA (FB-BirA) vectors were described in previous studies(87, 107). The pcDNA3.1(+)-RPS2-TEV-3×-FLAG (FL-RPS2)

12 J. Biol. Chem. (2022) 298(7) 102084

and pcDNA3.1(+)-RPL23A-TEV-3×-FLAG (FL-RPL23A) vectors were constructed in this study. The human RPS2
(NM_002952.3) and RPL23A (NM_000984.5) cDNAs were
ampliﬁed from HEK293T cells using a forward primer
including a BamH1 restriction site and a reverse primer
including a TEV protease site followed by the 3× FLAG
sequence and an Xho1 restriction site. The ampliﬁed cDNAs
were then introduced into the pcDNA 3.1 (+) vector at the
BamH1 and Xho1 sites. Therefore, the resulting plasmids
encode C-terminal 3× FLAG-tagged RPS2 and 3× FLAGtagged RPL23A. The MSCV-FB-eEF1A1 (FB-eEF1A1) and
MSCV-FB-eEF1A1K55R mutant (FB-eEF1A1K55R) plasmids
were constructed in this study. Human eEF1A1 cDNA
(NM_001402.5) was PCR-ampliﬁed using cDNA from theASPC1 cell line and was inserted into the MSCV vector using
its BamH1 restriction site. The MSCV-FB-eEF1A1K55R
mutant vector was then constructed by introducing the point
mutation to the MSCV-FB-eEF1A1 plasmid. To construct the
pcDNA-IMP4-His (His-IMP4) plasmid, human IMP4 cDNA
was PCR-ampliﬁed from the human brain cDNA library(Human Brain, Hypothalamus Marathon-Ready cDNA, No.
639329, Takara Bio). The ampliﬁed cDNA was then inserted
into the HindIII and SacII sites of the pcDNA 3.1/myc-His B
vector (V800-20; Thermoﬁsher Scientiﬁc).
Western blot analysis
Western blot analysis was performed as described in previous studies with modiﬁcations (78, 87). Brieﬂy, cells werelysed by 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 1%
SDS, 10% glycerol, 1% 2-mercaptoethanol and 0.02% bromophenol blue) containing protease inhibitors (0469315900,
cOmplete Mini EDTA-free Protease Inhibitor Cocktail Tablets, Roche) or by radioimmunoprecipitation assay buffer(50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%
NP-40, 0.5% Na-deoxycholate, 0.1% SDS and cOmplete protease inhibitors). For detection of phosphorylated proteins, thecell lysis buffer additionally contained a phosphatase inhibitor(04906837001, PhosSTOP, Roche). Protein amount wasquantiﬁed by either a protein assay kit (22660, Pierce 660 nm
protein assay reagent, Thermo Fisher Scientiﬁc) or SDS-PAGE
followed by CBB (50% methanol, 10% acetic acid and 0.1%
R-250 or G-250) staining and quantiﬁcation of stain densitywith an ImageJ software (version 1.53i). The extracted protein
was denatured by heating at 95  C for 5 min in the presence of
the SDS-PAGE sample buffer. The protein was separated by
SDS-PAGE in an 11% polyacrylamide gel (15% for SUnSET
and ribosomal proteins). The separated proteins were thentransferred to 0.45-μm PVDF membrane (IPVH00010,
Immobilon-P Transfer Membrane, Merck Millipore). Aftertransfer, the PVDF membranes were blocked in 3 to 5% skim
milk in TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl and
0.05% Tween 20) before incubation with antibodies. Whenphosphorylated proteins were examined, antibody reactions
were done in TBS-T containing 3% BSA. The imaging wasdone by a ChemiDoc MP imaging system (Bio-Rad Laboratories) using Clarity Western ECL Substrate (1705060, Bio-Rad

mTORC1-independent translation control by MAT2A and SAM
Laboratories). The information on antibodies is provided in
the “Supplementary Method” section.
Translation assay
The translation rate was examined by SUnSET (52). Cells
were grown to 70 to 80% conﬂuency and pulse-labeled with
20 mg/ml puromycin (P8833, Puromycin dihydrochloride,
Sigma-Aldrich) for 30 min at 37  C in a cell culture incubator.
In serum stimulation, cells were grown in serum-free DMEM
containing 4.5 g/l glucose (D5796, Sigma-Aldrich) for a periodof 24 h. Then, cells were returned into DMEM supplemented
with 10% FBS for 1 h or 3 h before pulse labeling with puromycin. Cells were lysed in radioimmunoprecipitation assay
buffer supplemented with the protease inhibitors. Around 1 to
1.5 μg of total clariﬁed lysate protein was loaded in a lane for
SDS-PAGE and subsequent Western blotting detection was
done by using an antipuromycin antibody.
Polysome analysis
Approximately 5 × 106 cells were seeded in 25 ml of media
in 15-cm cell culture dishes and were grown for 18 h. The
cells in the treatment group were then treated with 30 mM
cLEU (A48105-10G, Sigma-Aldrich) for 6 h, and the cells
were harvested 24 h after the seeding. The polysome wasstabilized by treating with 100 μg/ml CHX (06741–04,
Nacalai Tesque) for 10 min. The harvested cells were lysed in
550 μl of hypotonic lysis buffer (10 mM Hepes-KOH pH 7.9,
1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1% Triton X-100,
100 μg/ml CHX, 40 U/ml RNAsin, and cOmplete proteaseinhibitor). The lysate was cleared by centrifugation at 15,000g
for 10 min. A total of 500 μl of lysate containing around
150 μg of total RNA was layered on 10 to 50% sucrose
gradient in polysome buffer (20 mM Hepes-KOH pH 7.6,
5 mM MgCl2, 100 mM KCl, 1 mM DTT, and 100 μg/ml
CHX) and was centrifuged at 274,000g for 1.5 h at 4  C(40,000 rpm using an SW41Ti rotor, 331362, Beckman
Coulter). A Piston Gradient Fractionator (Biocomb) equippedwith a single path UV-1 optical unit (Biomini UV-monitor,
ATTO) and a chart recorder (Digital mini recorder, ATTO)
was used for sucrose fraction collection and polysome proﬁlegeneration as previously described (108).
RNA sequencing
The library was prepared using an MGIEasy RNA Directional Library Prep Set (MGI Tech Co, Ltd) following the
manufacturer’s manual. To examine the prepared libraryquality, the circularized DNA was prepared using an MGIEasy
circularization Kit (MGI Tech Co, Ltd) with the manufacturer’s guideline. After making DBA nanoball by a DNBSEQG400RS High-throughput Sequencing Kit (MGI Tech Co,Ltd), the sequencing was performed using DNBSEQ-G400.
The differential gene expression analysis was performed fortranscripts aligned to more than ten reads using estimateSizeFactors function, estimateDispersions function, and nbinomWaldTest function in R package DESeq2.

Puriﬁcation of FB-MAT2A and LC-MS/MS analysis
Two million stable expression cells were seeded in four 10cm dishes 24 h before the cell harvest. The harvested cells
were lysed in 1 ml of IP buffer (10 mM Tris-HCL pH 7.4,
150 mM NaCl, 1 mM EDTA, 0.3% NP-40, 2 mM DDT,cOmplete protease inhibitor, and PhosSTOP phosphataseinhibitor). The clariﬁed supernatant was used for afﬁnity
puriﬁcation with streptavidin-conjugated magnetic beads(Dynabeads M-280 Streptavidin, Thermo Fisher Scientiﬁc)(78, 107). Brieﬂy, the cleared lysate and 20 μl of Dynabeads
slurry were incubated with an end-to-end rotation at 4  C for
2 h. The protein-bound beads were washed, and the proteins
were eluted in the presence of biotin at 70  C for 15 min. The
eluted protein was processed for mass spectrometry according to the previously described protocol (109, 110). Brieﬂy,
the puriﬁed proteins were separated by SDS-PAGE ussing a 5
to 20% gradient gel (HON-052013; Oriental Instruments Co,Ltd) and were stained with CBB for visualization. Every lane
was divided into small gel pieces and the pieces were treatedwith 30% acetonitrile (ACN) for destaining and then with 50%
and 100% ACN for dehydration. The samples were treatedwith DTT and then with acrylamide for reduction andalkylation of cysteine side chains. The samples were digested
with 10 to 30 ng of trypsin (V5280, Trypsin Gold, Promega) indigestion buffer (50 mM ammonium bicarbonate and 10%
ACN) overnight at 37  C. The digested peptides were eluted
in a solution containing 75% ACN and 1% formic acid solution. The eluates were then concentrated in SpeedVac andwere analyzed in an Orbitrap Fusion mass spectrometer
connected with an Easy-nLC 1000 HPLC (Thermo Fisher
Scientiﬁc). The obtained raw MS/MS data were then converted to mgf ﬁles using a Proteome discoverer software(version 1.3.0.339, Thermo Fisher Scientiﬁc). The converted
ﬁles were submitted to the database search for peptide
identiﬁcation using a Mascot search engine (Matrix Science)
considering propionamide (Cys) as a ﬁxed modiﬁcation andacetyl (Protein N-term) and oxidation (Met) as variable
modiﬁcations. The precursor ion and MS/MS tolerances were
set to 5 ppm and 0.5 Da, respectively. A maximum of three
miscleavage was allowed without considering nonspeciﬁccleavage. The human proteins in Swissprot (Jan 2018) and ahomemade contaminant protein list were searched. The
number of proteins included in the searched database were
20,560 in total. The threshold of Mascot expectation value forsigniﬁcant peptide-spectral matches were set to 0.05. The
false discovery rates estimated by Mascot decoy search werereported as follows: 162 PSM in decoy database versus 6262 inreal database (2.63%) for Control IP and 163 versus 7169
(2.27%) for FLAG-Bio-MAT2A IP. The result from a single
experiment is shown.
Quantiﬁcation of rRNA
Ribosomal RNA was quantiﬁed by a 2100 Bioanalyzer Instrument (Agilent) using an Agilent RNA 6000 Pico kit (50671513, Agilent) following the manufacturer’s instruction.

J. Biol. Chem. (2022) 298(7) 102084

13

mTORC1-independent translation control by MAT2A and SAM
Northern blotting
Northern blotting of rRNA precursors was performed according to a previously described protocol (111). Brieﬂy, total
RNA was isolated from HeLa cells treated with either cLEU or
none by using an RNeasy Plus Mini kit (74134, Qiagen)following the protocol supplied by the manufacturer. Four
micrograms of the total RNA were separated by electrophoresis using a 1.2% formaldehyde-agarose denaturing gel in thepresence of 1× TT (30 mM Tricine and 30 mM triethanolamine) at 200 V for 120 min. The separated RNA was thentransferred to Hybond-N+ membrane (RPN303B, GE
Healthcare) by a capillary method in the presence of 20× SSC(3 M NaCl and 300 mM trisodium citrate dihydrate) for 18 h.
The RNA was then UV-crosslinked at 120 mJ/cm2 by using a
CL-1000 UV-crosslinker device. The membranes were incubated in the hybridization buffer containing the DIG-labeled
hybridization probes at 50  C for 20 h. The membrane wasthen washed once in 2× SSC containing 0.1% SDS for 15 min
at 50  C, then twice in 0.1× SSC containing 0.1% SDS for twotimes at the same temperature, and ﬁnally in 1× MA buffer(100 mM maleic acid and 150 mM NaCl, pH 7.0) for 10 min atroom temperature. After being blocked by a blocking reagent(11096176001, Roche) for 30 min, the membrane was incubated with anti-digoxigenin–AP, Fab fragments (11093274910,
Roche) in the blocking buffer for 1 h. After that, the membrane
was washed three times in 1× MA buffer containing 0.3%
Tween-20 and then was equilibrated in buffer-A (100 mM
Tris-HCl and 100 mM NaCl, pH 9.5). RNA was detected using
CDP-star (11759051001, Roche) and a LAS-4000 mini device.
The 50 DIG-labeled probes for the detection of the precursor
rRNAs are follows:
ITS1: 50 -CCTCGCCCTCCGGGCTCCGGGCTCCGTTAATGATC-30
ITS2: 50 -CTGCGAGGGAACCCCCAGCCGCGCA-30
The mature human 18S and 28S rRNAs were detected by
DIG-labeled probes that were synthesized by PCR DIG Probe
Synthesis Kit (11636090910, Roche) using the following
primers.
18S rRNA: 50 -ATCAAGAACGAAAGTCGGAGGTTCG-30
and 50 -GTGCAGCCCCGGACATCTAAG-30 .
28S rRNA: 50 -GCCGACTTAGAACTGGTGCGG-30 and
0
5 -CTCACCGGGTCAGTGAAAAAACGA-30 .
Heavy-methyl SILAC experiment
The heavy methyl-SILAC experiment was conducted basedon the principles and procedure described in earlier researches(68, 112, 113). The SILAC labeling media were prepared byreconstituting commercially available DMEM high-glucose
media depleted for glutamine, methionine, and cysteine(21013024; DMEM high glucose, no glutamine, no methionine, no cysteine cell culture media, Thermo Fisher scientiﬁc).
For reconstitution, we added 4 mM glutamine (25030-081,
Thermo Fisher scientiﬁc), 200 μM cysteine (1001527621,
L-cysteine dihydrochloride, Sigma-Aldrich) and either 200 μM
normal methionine (1001818815; L-Methionine, SigmaAldrich) for the light medium or 200 μM heavy methionine

14 J. Biol. Chem. (2022) 298(7) 102084(CDLM9289025, L-methionine, Methyl-13C 99%, Methyl-D3
98%, Cambridge Isotope Laboratories) for the heavy medium. After reconstitution, we added 10% dialyzed FBS (F0392,
Sigma-Aldrich) and a combination of 100 U/ml penicillin and100 μg/ml streptomycin (15140-122, Penicillin-Streptomycin,
Thermo Fisher scientiﬁc). HeLa cells were cultured in either
light or heavy medium for at least six generations. The heavylabeled cells were treated with 30 mM cLEU for 6 h before
harvest. The ribosome was puriﬁed as follows:
Method 1 (puriﬁcation by IP)
Light- and heavy-labeled cells were transfected with 15 μg ofeither the FL-EV or FL-RPL23A plasmid and were grown for
24 h before harvest. After cell harvest by trypsinization, an
equal number of light- and heavy-labeled cells were mixed andwere lysed in 700 μl of lysis buffer (10 mM Hepes-KOH pH
7.4, 100 mM NaCl, 10 mM MgCl2, 10% glycerol, 2 mM DTT,
1 mM PMSF, 2% NP-40, and cOmplete protease inhibitor).
The cleared lysate was then used for ribosome puriﬁcation by
an anti-DDDDK antibody bound to magnetic beads (M18511R, anti-DDDDK-tag mAb-magnetic beads, MBL) at 4  C for
2 h. The ribosomes were then heat-eluted and were denatured
in the presence of SDS-sample buffer.
Method 2 (puriﬁcation by pelleting on sucrose cushion)
5 × 106 of light- and heavy-labeled cells were mixed in 1:1
ratio and were lysed in 350 μl of lysis buffer (20 mM Tris-HCL
pH 7.5, 150 mM NaCl, 5 mM MgCl, 1 mM DTT, 1% TritonX100, cOmplete protease inhibitor, and100 μg/ml CHX). The
lysate was clariﬁed by centrifugation at 2000g for 20 min at 4

C. The cleared lysate was then layered on 900 μl of 1 M sucrose cushion in a 13 × 56 mm 3.2-ml capacity thick-wallpolycarbonate ultracentrifuge tube (362305; Beckman
Coulter). The tube was then centrifuged in a TLA-110 ﬁxed
angle rotor (366735, Beckman Coulter) at 543,000g(100,000 rpm) for 1 h at 4  C in an Optima MAX-XP Benchtop
Ultracentrifuge (Beckman Coulter). The resulting ribosome
pellet was washed with ice-cold PBS, was dissolved in SDSsample buffer and was denatured at 95  C for 5 min.
After puriﬁcation of ribosome by either method, the LCMS/MS sample preparation and analysis were performed as
described in “Puriﬁcation of FB-MAT2A and LC-MS/MS
analysis” except that methylation and demethylation at Lys
and Arg and trimethylation at Lys were also included in thevariable modiﬁcations. The human proteins in Swissprot (Nov
2020) and a homemade contaminant protein list weresearched. The number of proteins included in the searched
database was 20,692 in total. The threshold of Mascot expectation value for signiﬁcant peptide-spectral matches was set to0.05. The false discovery rates estimated by Mascot decoy
search were reported as follows: 118 PSM in decoy database
versus 12,888 in real database (0.92%) for ribosome isolation by
ultracentrifugation and 65 versus 5025 (1.29%) for FLAGRPL23a IP. The result from a single experiment is shown.
Details in the methylated peptides identiﬁcation procedure canbe found in “Supplementary Method” section.

mTORC1-independent translation control by MAT2A and SAM
Statistical analysis
Welch two-sample t test was performed using an R program(Version 4.0.2) for comparison of Mat2a mRNA (Fig. S1A) andrRNA ratio (Fig. 5B). The relative intensity values wereanalyzed by one-way analysis of variance assuming equalvariance followed by Tukey’s honest signiﬁcance test for thecomparison of multiple means (Fig. 1B). Two sample unpairedt test was performed to compare the normalized intensities ofpuromycin-labeled peptides (Fig. 1C). GO analysis of proteinsor genes were performed by using package ‘Go.db” with“enrichGo” function in R. Pearson correlation analysis was
performed to compare the correlation between the abundanceof light and heavy unmethylated methionine-containing peptides (Fig. 6, C and D) using package “ggpmic” with “stat(eq.label)” function in R.

Data availability
The raw data ﬁles of mass spectrometry are available in the
MassIVE
publicdatabase(https://massive.ucsd.edu/
ProteoSAFe/static/massive.jsp, Dataset ID: MSV000089292
and MSV000089296), and the raw data for mRNA sequencing
are available in GEO with ID: GSE201299.
Supporting information—This article contains supporting information (59, 87, 88, 114).
Acknowledgments—We are thankful to Dr Tadashi Nakagawa,
Department of Cell Proliferation, Tohoku University Graduate
School of Medicine, for the technical advice on the SUnSET
experiment. We are grateful to the members of our laboratory for
their useful suggestions and advice. We would like to express our
gratitude also to Moeka Seki, Laboratory of Gene Regulation,
Tohoku University Graduate School of Pharmaceutical Science
for the supply of HeLa cells during the research works. We arealso grateful to Dr Kenta Teruya, Associate Professor of the
Department of Neurochemistry, Tohoku University Graduate
School of Medicine for supplying human cDNA library during
gene cloning.
Author contributions—M. A., H. S., Y. M., N. C. L., T. S., N. S., R. N.,
S. H., L. L., M. K. K., and Y. K. investigation; M. A. writing-originaldraft; H. S., K. I., and T. I. supervision; H. S., K. I., and T. I. writingreview and editing; N. C. L., M. M., T. I., and K. I. formal analysis; Y.
I. and T. S. resources.
Funding and additional information—This study has been supported by Grants-in-Aid for Scientiﬁc Research from the Japan
Society for the Promotion of Science (22H00443, 20KK0176 and
18H04021 to K. I., 18H03977 to T. I., 20K07321 to H. S., and19K07680 and 16K07108 to M. M.), Research Grant in the Natural
Sciences from the Mitsubishi Foundation (to K. I.), AMED underGrant Number JP19gm1110010 (T. I.) and Platform Project for
Supporting Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science Research[BINDS]) from AMED under Grant Numbers JP21am0101078 andJP22ama121008 (to Y. K.).
Conﬂict of interest—The authors declare that they have no conﬂicts
of interest with the contents of this article.

Abbreviations—The abbreviations used are: 4E-BP, 4E binding
protein; ACN, acetonitrile; CBB, Coomassie Brilliant Blue; CHX,
cycloheximide; cLEU, cycloleucine; DMEM, Dulbecco’s Modiﬁed
Eagle’s Medium; eIF4A, eukaryotic initiation factor 4A; eIF4B,
eukaryotic initiation factor 4B; FB-MAT2A, FLAG-Bio-tagged
MAT2A; FBS, fetal bovine serum; IP, immunoprecipitation; ITSs,
internal transcribed spacers; MAT, methionine adenosyltransferase;
mTORC1, mechanistic target of rapamycin complex 1; S6K, S6
kinase; SAM, S-adenosylmethionine; SILAC, stable isotope labelingby amino acid in cell culture; TE, translation efﬁciency.

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