Letter reSeArCH
m^6 A can be enriched in stress granules (Fig. 4b), which suggests that
mRNAs can also be recruited through m^6 A-independent mechanisms.
It is possible that phase partitioning of DF–m^6 A-mRNA complexes
to different phase-separated compartments would impart a different
fate to m^6 A-mRNAs. In the case of unstressed cells, the targeting of
DF2 and m^6 A-mRNA to P-bodies facilitates the degradation of m^6 A-
mRNA^25. We therefore asked whether the relocalization of DF proteins
and m^6 A-mRNA to stress granules affects mRNA abundance. Cellular
mRNA levels were examined using RNA sequencing in wild-type mES
cells before heat shock, immediately after heat shock, and after a one-
hour recovery period. The results showed no substantial alteration
in the expression of m^6 A-modifed mRNAs (Fig. 4e, Extended Data
Fig. 5a, Supplementary Table 1), which suggests that DF–m^6 A-mRNA
complexes in stress granules do not induce mRNA degradation.
We used ribosome profiling to compare the translation efficiency of
mRNAs when they contain m^6 A (in wild-type cells) relative to when
they lack m^6 A (in Mettl14-knockout cells). In this way, we can deter-
mine the effect of m^6 A on each mRNA in the dataset. Before heat shock,
we found no substantial effect of m^6 A on translation efficiency (Fig. 4f,
Supplementary Table 2). As expected, ribosome-protected fragments
were markedly reduced after 30 min of heat shock, which is consistent
with the global translational suppression reported during mosta c
Total Stress granuleControl Heat shock Arsenite00.51.01.52.0Relative fold changem
6 A/(A+C+U)efmES cells, 30 min heat shock
bNS****dm^6 A peaks per gene
0 (14,051)
1 (3,109)
2 (1,826)
3 (914)
4+ (668)(no. of genes)
Cumulative probability1.000.750.500.250.00
–4 –3 –2–1^02314
Fold change, log 2 (stress granule mRNA/total mRNA)ArseniteGrk6 Fignl10.00.20.40.60.8Fraction smFISH in stress granulesPolr2a Fem1bNon-methylated mRNA
Polymethylated mRNA
(P = 0.0075) (P = 0.0068)TIAR Grk6 mRNA Fignl1 mRNAMergeTIAR Polr2a mRNAFem1b mRNAMerge0 1 2 3
Fold change, log 2 (WT, heat shock/no stress)Cumulative probability0.501.00
0
2–3
4+1m^6 A per gene
0.750.250.00
–3–2 –1No stress0 1 2–3 4+
Annotated m^6 A sites per geneFold change, log(translation 2ef ciency, WT/Mettl14KO)01.0–1.0
–2.03.0–3.0n = 3,361n = 1,591n = 993n = 325NS (P = 0.30)NS (P = 0.19)2.0 NS (P = 0.43)1-h recovery01.0–1.0
–2.02.00 1 2–3 4+
Annotated m^6 A sites per gene3.0–3.0********
NS (P = 0.21)n = 3,762
n = 1,816n = 1,168n = 385Fold change, log(translation 2ef ciency, WT/Mettl14KO)gpoly(A) RNA poly(A) RNA(P = 0.46)(P = 0.005)(P = 0.008)** **Fig. 4 | m^6 A-containing mRNAs are enriched in distinct DF-containing
RNA granules. a, m^6 A levels were measured in poly(A) RNA purified
from the insoluble stress-granule-enriched fraction and poly(A) RNA
prepared from total NIH3T3 cellular extracts. m^6 A levels were quantified
by thin-layer chromatography, and normalized to the combined intensities
of A, C and U. In non-stressed cells, there was no significant difference in
the levels of m^6 A-mRNA in the total cellular or insoluble RNA fraction.
By contrast, a significant increase in m^6 A levels was detected in the stress-
granule fraction obtained from either heat-shocked or arsenite-stressed
cells (control, n = 3; heat shock, n = 4; arsenite, n = 4; where n represents
biological replicates). Bar heights represent mean normalized fold change
of m^6 A/(A+C+U) in poly(A) RNA from stress granules compared
with the poly(A) RNA from total cellular RNA (control = 1.076, heat
shock = 1.49, arsenite = 1.503). Filled circles and diamonds with
lines represent paired biological samples. Error bars represent s.e.m.
Paired two-sided Student’s t-test was performed on unnormalized
m^6 A/(A+C+U) fractions between control and stress conditions.
b, A cumulative distribution plot of mRNA enrichment in U2OS arsenite-
induced stress granules^23 was plotted for mRNAs classified by the number
of annotated m^6 A peaks^31 per transcript. Transcripts with no m^6 A peaks
(that is, non-methylated) are slightly depleted in stress granules relative
to total cellular RNA. However, transcripts that contain two or more m^6 A
peaks show enrichment in stress granules in proportion to the number
of m^6 A sites. c, d, m^6 A-containing mRNAs show higher enrichment
in stress granules compared to non-methylated mRNAs, as visualized
using smFISH (c). Two mRNAs that lack annotated m^6 A sites (Grk6 and
Polr2a) were matched with m^6 A-containing mRNAs of similar length and
abundance (Fignl1 and Fem1b, four m^6 A sites each^32 ). Grk6 and Polr2a are
not enriched in stress granules (d). Fignl1 and Fem1b are markedly more
enriched within stress granules as a fraction of total smFISH puncta
after heat-shock stress. In c, r epresentative slices from confocal Z-stacks
are shown to demonstrate localization. In d, images (Grk6/Fignl1:
n = 5 images, 26 cells, 2 biological replicates; Polr2a/Fem1b: n = 5
images, 24 cells, 2 biological replicates) were analysed to assess mRNA
localization to stress granules. Bar heights represent mean fraction of
stress-granule-localized smFISH puncta and error bars represent s.e.m.
Two-sided Student’s t-test. e, mRNA expression levels were determined by
RNA sequencing before and after heat shock (42 °C, 30 min). Transcript
abundance was unaltered for non-methylated, singly methylated and
polymethylated m^6 A-mRNAs. f, Translation efficiency before heat shock
was calculated using matched ribosome-profiling and RNA sequencing
data and compared for each mRNA in the methylated state (that is, in
wild-type cells) versus the non-methylated state (that is, in Mettl14-
knockout cells). Transcripts were binned on the basis of the annotated
number of m^6 A sites as in e. The centre of the box plot represents the
median log 2 -transformed fold change, the boundaries contain genes
within a quartile of the median, and the whiskers represent genes in the
upper and lower quartiles. m^6 A-mRNAs in wild-type mES cells did not
show a significant difference in translational efficiency compared to
Mettl14-knockout mES cells. n denotes the number of genes in each
bin. Binned gene groups with annotated m^6 A sites were compared to
genes with no m^6 A sites with an unpaired two-sided Student’s t-test.
g, Translation efficiency in wild-type and Mettl14-knockout mES cells
subjected to 30 min of continuous heat shock (42 °C, 30 min) followed
by 1 h recovery at 37 °C. Only polymethylated transcripts showed
significantly decreased translation efficiency. The effect of m^6 A was
determined by comparing the translation efficiency for each transcript in
the methylated form (wild-type cells) relative to the same transcript in the
non-methylated form (Mettl14-knockout cells). The same binning and
m^6 A annotation strategy were used as in f. Boxplots are presented as in f.
n denotes the number of genes in each bin (total number of genes: 6,720
(f) and 7,131 (g)). Binned gene groups with annotated m^6 A sites were
compared to genes with no m^6 A sites with an unpaired two-sided Student’s
t-test. ****P < 0.0001.18 JULY 2019 | VOL 571 | NAtUre | 427