RNA Chemical Proteomics Reveals the N6-Methyladenosine (m6A)-Regulated Protein-RNA Interactome

Article abstract of DOI:10.1021/jacs.7b09213

Epitranscriptomic RNA modifications can regulate mRNA function; however, there is a major gap in our understanding of the biochemical mechanisms mediating their effects. Here, we develop a chemical proteomics approach relying upon photo-cross-linking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N⁶-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic RNA. In addition to identifying YTH domain-containing proteins and ALKBH5, known interactors of this modification, we find that FMR1 and LRPPRC, two proteins associated with human disease, "read" this modification. Surprisingly, we also find that m⁶A disrupts RNA binding by the stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42. Our work provides a general strategy for interrogating the interactome of RNA modifications and reveals the biochemical mechanisms underlying m⁶A function in the cell.

Full text of DOI:10.1021/jacs.7b09213

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Communication
An RNA chemical proteomics approach reveals the N6-
methyladenosine (m6A)-regulated protein-RNA interactome
A. Emilia Arguello, Amanda N. DeLiberto, and Ralph Kleiner
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09213 • Publication Date (Web): 15 Nov 2017
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An RNA chemical proteomics approach reveals the N -
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methyladenosine (m A)-regulated protein-RNA interactome
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A. Emilia Arguello, Amanda N. DeLiberto, & Ralph E. Kleiner*
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
Supporting Information Placeholder
stability, localization, and translation. Indeed, stud-
ies of m A have demonstrated how recognition of
this modification by YTH-domain containing pro-
teins is involved in RNA metabolism , protein trans-
lation , and splicing . These findings suggest a gen-
eral mechanism underlying the function of epitran-
scriptomic modifications; however, currently we lack
ABSTRACT: Epitranscriptomic RNA modifications
can regulate mRNA function; however, there is a ma-
jor gap in our understanding of the biochemical
mechanisms mediating their effects. Here, we devel-
op a chemical proteomics approach relying upon
6
6
7
8
photo-crosslinking
with
synthetic diazirine-
containing RNA probes and quantitative proteomics
6
6
a comprehensive understanding of how m A, as well
to profile RNA-protein interactions regulated by N -
6
as other epitranscriptomic modifications, affect the
global landscape of cellular protein-RNA interac-
tions.
methyladenosine (m A), the most abundant internal
modification in eukaryotic RNA. In addition to iden-
tifying YTH domain-containing proteins and
ALKBH5, known interactors of this modification, we
find that FMR1 and LRPPRC, two proteins associated
with human disease, ‘read’ this modification. Sur-
Identifying direct protein-RNA interactions is
challenging since interactions can be low affinity and
protein complexes make it difficult to distinguish
direct from indirect binders. In addition, evaluating
interactions mediated by modified RNA requires
targeted incorporation or enrichment of the modifi-
cation. Photo-crosslinking strategies have been
widely used to stabilize direct protein-RNA interac-
6
prisingly, we also find that m A disrupts RNA bind-
ing by the stress granule proteins G3BP1/2, USP10,
CAPRIN1, and RBM42. Our work provides a general
strategy for interrogating the interactome of RNA
modifications and reveals the biochemical mecha-
6
9-10
nisms underlying m A function in the cell.
tions . These approaches rely on the propensity of
natural nucleobases or of sulfur- or halogen-
containing nucleobase derivatives for UV-induced
photochemistry. In contrast, photo-affinity labels
such as diazirine or benzophenone , which can be
excited at longer wavelengths and may offer higher
efficiency crosslinking, have not seen widespread use
in the interrogation of protein-RNA interactions.
Chemical modifications on biological macromole-
cules play a critical role in regulating their function.
RNA is extensively modified by cellular enzymes and
contains over 100 different post-transcriptional mod-
11
12
1
ifications . While most modifications are found in
tRNA, recently, a new class of RNA modifications has
been identified. These ‘epitranscriptomic’ modifica-
Here, we develop a chemical proteomics approach,
relying upon a high-efficiency diazirine-based RNA
photo-crosslinker and quantitative proteomics, to
profile m A-regulated RNA-protein interactions. We
identify known m A binders, thereby validating our
2
6
tions , best exemplified by N -methyladenosine
(
6
3-4
6
m A) , occur on both internal mRNA sequences
6
and non-coding RNA and are postulated to regulate
gene expression. Our current understanding of how
these modifications affect RNA function and their
role in biological processes is still limited.
6
method. In addition, we find new ‘readers’ of m A
and several proteins that are repelled by this modifi-
cation. Taken together, our study demonstrates how
photo-crosslinking and proteomics can be combined
to profile the interactome of modified RNA and ex-
The proper regulation of mRNA behavior relies on
interactions with a large complement of RNA-
5
binding proteins (RBPs) that control RNA splicing,
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pands the known repertoire of m A-regulated pro-
tein-RNA interactions.
Figure 1. RNA photo-affinity probes for studying
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m A-dependent protein-RNA interactions. (a) Struc-
tures of RNA probes used in this work. (b) Photo-
crosslinking of YTHDC1-YTH and probes 1, 3, or 4.
Protein was mixed with various concentrations of
probe and UV irradiated. Streptavidin WB was used
to detect product formation (see Supplementary Fig.
1 for full WB). Values represent mean +/- s. d. (n = 3).
Asterisks represent statistically significant differ-
ences in cross-linking between probes 1 and 3, and
probes 3 and 4 (*: p < 0.05; **: p < 0.005) (c) Photo-
crosslinking of YTHDC1-YTH with probes 1 or 2. Ex-
periment was performed as in (b) and EC50 values
were determined by fitting data to a sigmoidal dose-
response curve. Values represent mean +/- s. d.
(probe 1, n = 6; probe 2, n = 3) (see Supplementary
Fig. 3 for full WB). (d) Competition of photo-
6
To profile ‘readers’ of m A using photo-
crosslinking-based affinity isolation and quantitative
proteomics, we needed RNA probes containing: i)
m A, ii) an efficient photo-crosslinker that does not
interfere with protein-RNA interactions, and iii) an
6
affinity handle for protein enrich-
a
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b
N
“
m A” probe
N
6
AꢀCꢀ5ꢀDzUꢀGUACꢀbiotin
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probe 1: GGꢀm
5
ꢀDzU
O
4ꢀSU
S
5ꢀIU
O
CH
3
O
NH
HN
O
O
O
S
NH
NH
I
NH
N
N
HN
N
N
H
N
N
HN
NH
N
O
N
O
N
O
N
O
N
probe 1
.04
probe 3
0.04
probe 4
0
1
1
0.04
1 ꢁM
YTHDC1ꢀ
YTH
“A” probe
probe 2: GGACꢀ5ꢀDzUꢀGUACꢀbiotin
WB: streptavidin
1
00
NH
2
O
O
probe 1
probe 3
probe 4
O
S
N
N
HN
N
80
N
H
N
N
HN
NH
N
O
N
6
0
0
4
6
20
0
**
**
probe 3: GGꢀm AꢀCꢀ4ꢀSUꢀGUACꢀbiotin
*
crosslinking between YTHDC1-YTH and probe 1
*
*
**
**
probe 4: GGꢀm
6
AꢀCꢀ5ꢀIUꢀGUACꢀbiotin
0
.037
0.11
0.33
1
6
[RNA probe] (ꢁM)
with
5’-GAACCGG-m A-CUGUCUUA
or
5’-
c
6
d
GGACU
competitor
probe 1 (GGm ACU) probe 2 (GGACU)
6
GGm ACU competitor
GAACCGGACUGUCUUA. Experiment was per-
formed as in (b) and (c) using 0.1 µM protein, 0.1 µM
probe 1 and various concentrations of competitor
RNA. Values represent mean +/- s. d. (n = 4) (see
Supplementary Fig. 5 for full WB).
0.001
1
0.001
1 ꢁM
0.005
30
3.3 10 30 ꢁM
YTHDC1ꢀ
YTH
YTHDC1ꢀ
YTH
WB: streptavidin
WB: streptavidin
120
00
1
20
B
J
probe 1: EC50 = 46 +/ꢀ 14 nM
probe 2: EC50 > 1 ꢁM
IC50 = 0.54 +/ꢀ 0.24 ꢁM
1
B
B
100
80
60
40
20
0
B
B
8
0
0
0
0
0
B
B
B
13-14
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4
2
ment. Guided by available structural , biochemi-
B
B
6, 13-14
3-4
B
cal
, and transcriptomic data , we prepared
J
B
6
B
J
B
J
B
probe 1 (Fig. 1a), which contains the consensus m A
site in mammalian cells, GGm ACU. This sequence is
sufficient for binding to YTH-domain proteins
We replaced uridine in the GGm ACU motif with a
photo-crosslinkable diazirine-modified uridine resi-
due (5-DzU) and incorporated biotin at the 3’ end of
B
B
J
J
0.001
0.01
0.1
1
0.001
0.01
0.1
1
10
100
6
[probe] (ꢁM)
[GGm ACU competitor] (ꢁM)
6
13-14
.
6
the probe. In addition, we synthesized probe 2 (Fig.
6
1
a), which contains A instead of m A, but is other-
wise identical to probe 1, and probes 3 and 4 (Fig. 1a),
6
which contain m A flanked by the photo-crosslinker
4-thiouridine (4-SU) or 5-iodouridine (5-IU), respec-
tively.
To determine the optimal photo-crosslinker, we as-
6
sayed photo-crosslinking between m A-containing
probes 1, 3, and 4 and purified YTH domain from
YTHDC1 (hereafter ‘YTHDC1-YTH’). For each probe,
we observed a dose-dependent increase in photo-
crosslinking in the 0.037 to 1 µM concentration re-
gime (Figure 1b and Supplementary Fig. 1); however,
we observed significantly greater product formation
using the 5-DzU probe (1) than for either the 4-SU
(3) or 5-IU (4) probe. Quantification of photo-
crosslinking yield indicated that the 5-DzU probe (1)
generated 6-fold more product than the 4-SU probe
(3) and 30-fold more than the 5-IU probe (4) (Fig.
1
b). A similar trend in probe reactivity was observed
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for the YTH domain from YTHDF2 (hereafter
ine-containing probes 1 and 2 effectively recapitulate
the affinity and specificity of known m A readers.
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‘
YTHDF2-YTH’) (Supplementary Fig. 2).
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Due to its high photo-crosslinking efficiency, we
We next applied our probes to identify m A read-
further characterized probe 1 as well as the corre-
sponding unmethylated probe (2) to confirm that
they recapitulated the interactions between YTH-
ers in cellular lysate. We set up comparative prote-
omics experiments in which HeLa cell lysate was
photo-crosslinked with 1 µM probe 1 or 2. Cross-
linked complexes were then isolated by streptavidin
enrichment, eluted from the bead with RNase treat-
6
domain proteins and native m A-modified or un-
methylated RNA. We setup photo-crosslinking titra-
tions between probes 1 and 2 and YTHDC1-YTH or
YTHDF2-YTH and observed dose-dependent photo-
crosslinking with probe 1 (YTHDC1-YTH, EC50 = 46
15
ment , and analyzed by LC-MS/MS to quantify pro-
0
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tein abundance (Fig. 2a). We performed 3 independ-
ent biological replicates of each proteomics experi-
ment to reliably quantify differences in photo-
crosslinking between probes 1 and 2, and a ‘volcano
plot’ was used to display the results (Fig. 2b, Sup-
plementary Datafile).
+
/- 14 nM; YTHDF2-YTH, EC50 = 0.42 +/- 0.12 µM)
(Fig. 1c, Supplementary Fig. 3 and 4) while photo-
crosslinking with probe 2 proceeded less efficiently
(Fig. 1c). We also showed that photo-crosslinking
between probe 1 and both YTH-domain proteins can
Our analysis of proteins specifically enriched by
6
be inhibited by adding an m A-competitor sequence
probe 1 (Fig. 2b, top right) identified several known
6
7
6
(YTHDC1-YTH, IC50 = 0.54 +/- 0.24 µM; YTHDF2-
‘readers’ of m A RNA including YTHDF1 , YTHDF2 ,
3, 16
8, 13
YTH, IC50 = 4.03 +/- 1.86 µM) (Fig. 1d, Supplemen-
tary Fig. 5 and 6), while competition with the identi-
cal unmethylated sequence was less efficient, con-
sistent with the reported affinity of these proteins for
YTHDF3 , and YTHDC1 , which showed between
- to 37-fold preference for the m A probe (1). We did
6
8
identify the fifth member of the mammalian YTH-
domain protein family, YTHDC2, enriched against
probe 1 (but not probe 2) in one
6
13-14
both GGm ACU and GGACU RNA sequences
.
Taken together, our results demonstrate that diazir-
a
b
6
6
GGm ACU binders
GGACU binders
GGACU binders
RBM42
GGm ACU binders
4.5
.0
3.5
LUC7L3
CAPRIN1
YTHDF3 YTHDF1
4
G3BP2 G3BP1
LRPPRC YTHDC1
YTHDF2
USP10
probe 1
GGm ACU)
probe 2
(GGACU)
6
hυ
hυ
(
SNRPB
CH
3
HN
N
NH
N
2
3
2
.0
.5
N
N
N
O
S
N
N
O
S
N
HN
NH
HN
NH
MEX3B
streptavidin
pulldown
streptavidin
pulldown
2.0
FMR1
1
1
0
.5
.0
.5
0
SAMHD1
ALKBH5
p < 0.05
RNase
elution
RNase
elution
LCꢀMS/MS
LCꢀMS/MS
ꢀ
6
ꢀ4
ꢀ2
0
2
4
6
6
m/z
m/z
log (m A/A spectral counts)
2
6
Figure 2. Proteomic profiling of the m A interactome. (a) Schematic for comparative proteomics workflow. (b) Vol-
6
cano plot of protein enrichment ratios (m A/A spectral counts) and p-values from proteomics experiments (n = 3)
(
see Supplementary Datafile). To calculate enrichment ratios for proteins identified by only one probe, we added 1 to
all spectral count values. P-values < 0.0001 were approximated as 0.0001.
biological replicate (Supplementary Datafile), alt-
hough the lack of reproducibility in this data pre-
cluded reliable quantification of binding prefer-
ence. In addition to YTH proteins, we also found
this protein may bind less tightly or is less abun-
dant in HeLa cells.
6
We also identified several novel binders to m A
RNA, including LRPPRC, and fragile X-related pro-
6
17
19
ALKBH5 (Fig. 2b), a known m A demethylase ,
teins FMR1 and FXR2, displaying between 2-3-fold
preference for probe 1 (Fig. 2b, Supplementary Da-
tafile). We validated the interaction between
exhibiting 2.4-fold preference for probe 1 over
18
probe 2. Interestingly, we failed to identify FTO ,
6
the other major m A demethylase, suggesting that
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LRPPRC and probe 1 by photo-crosslinking with 4-10-fold over AAACU and 60-100-fold over GUACU
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lysate generated from 293T cells expressing FLAG-
tagged LRPPRC. Gratifyingly we observed preferen-
tial crosslinking of LRPPRC to the m A probe (1)
(Fig. 3f and Supplementary Fig. 15).
Here, we have developed a chemical proteomics
approach to profile cellular proteins that bind to
6
6
over the unmethylated probe (2) (Fig. 3a and Sup-
m A-
plementary Fig. 8). In addition, we generated RNA
6
probes containing m A (7) or A (8) (Supplemental
Table 1) within the GUACU sequence context and
assayed them for photo-crosslinking to LRPPRC.
Interestingly, LRPPRC cross-linked preferentially to
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the m A-containing probe (7) (Fig. 3a and Supple-
mentary Fig. 8) within this different sequence
6
showing that recognition of m A does not depend
strongly on adjacent nucleotide identity. We also
investigated the putative interaction between FMR1
6
and GGm ACU RNA. We generated recombinant
FMR1 and assayed it for photo-crosslinking to
6
probe 1 in the presence of an m A-competitor or
unmethylated RNA sequence. Using this assay, we
observed a significant 2-fold preference in binding
6
of FMR1 to m A-RNA (IC = 3. 3 +/- 1.2 µM) over
50
unmethylated RNA (IC50 = 6.8 +/- 2.5 µM) (Figure
3b, Supplementary Fig. 9 and 10).
Unexpectedly, we also identified proteins en-
riched by the unmethylated probe (2) (Fig. 2b, top
left). Several of these proteins are found in stress
20
21
granules , including G3BP1, G3BP2, RBM42 ,
USP10, and CAPRIN1, all exhibiting between to 2.2-
to 25-fold preference for probe 2. G3BP1 has been
Figure 3. Characterization of novel protein-RNA in-
teractions. (a) Photo-crosslinking of LRPPRC to
probes 1, 2, 7, and 8. Lysate generated from 293T cells
transfected with 3x-FLAG-LRPPRC was photo-
crosslinked with 1 µM probe and streptavidin enriched
protein-RNA complexes were detected by anti-FLAG
WB (see Supplementary Fig. 8 for full WB data and
statistical analysis). (b) Competition of photo-
crosslinking between FMR1 and probe 1. Reactions
were performed and analyzed as in Fig. 1d but with
22
reported to interact with USP10 and CAPRIN1 ,
suggesting that these proteins may be involved in
related signaling pathways. We investigated the
interaction of these proteins with GGACU RNA by
performing photo-crosslinking reactions using
HeLa cell lysate or lysate from HEK293T cells ex-
pressing a FLAG-tagged transgene. Our results
show that USP10, CAPRIN1, and RBM42 all interact
preferentially with probe 2 (Fig. 3c and 3d, Supple-
mentary Fig. 11, 12, 13, 14, and 15), as predicted by
our mass spectrometry data.
200 nM GST-FMR1 and 1 µM probe 1. Values represent
mean +/- s. d. (n = 7) (see Supplementary Fig. 8 for
WB data). Asterisk represents statistically significant
differences (p < 0.05). (c) Photo-crosslinking of USP10
and CAPRIN1 to probes 1 and 2. Experiments were
performed as in (a). (see Supplementary Fig. 11 and 12
for full WB data and statistical analysis; **: p < 0.005;
For G3BP1, we generated recombinant protein
and performed photo-crosslinking titrations with
probes 1 and 2. We measured an EC of 240 +/- 42
5
0
*
: p < 0.05) . (d) Photo-crosslinking of RBM42 to
nM for the reaction between the GGACU probe (2)
and G3BP1 (Fig. 3e and Supplementary Fig. 14). In
contrast, photo-crosslinking between G3BP1 and
probes 1 and 2. HeLa cell lysate was photo-crosslinked
with 1 µM probe and streptavidin enriched complexes
were detected by anti-RBM42 WB (see Supplementary
Fig. 13 for full WB data and statistical analysis; *: p <
0.05). (e) Photo-crosslinking of recombinant G3BP1
and probes 1 or 2. Experiment was performed as in
Fig. 1c. Values represent mean +/- s. d. (n = 3) (see
Supplementary Fig. 14 for full WB. (f) Photo-
crosslinking of recombinant G3BP1 with unmethylat-
ed probes 2, 6, and 8. Experiment was performed and
6
the m A probe (1) proceeded much less efficiently
(
EC > 3 µM), consistent with our mass spectrome-
50
try data. We also assayed photo-crosslinking be-
tween G3BP1 and related unmethylated probes con-
taining the sequence AAACU (6) or GUACU (8)
and found that the GGACU motif was preferred by
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for molecular biology assistance. R.E.K. is a Dale F.
analyzed as in Fig. 1b (see Supplementary Fig. 15 for
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5
6
full WB). Asterisks represent statistically significant
differences in cross-linking between probes 2 and 6,
and probes 6 and 8 (*: p < 0.05; **: p < 0.005).
Frey Breakthrough Scientist of the Damon Runyon
Cancer Research Foundation (DFS #21-16) and a Sid-
ney Kimmel Foundation Scholar. All authors thank
Princeton University for financial support.
modified RNA. We identified YTH-domain pro-
teins and ALKBH5, known ‘readers’ and ‘erasers’ of
this mark, thereby validating our method. In addi-
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gous to the known functions of YTH domain-
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containing proteins. Indeed, both FMR1
and
2
25
LRPPRC are involved in translational regulation.
Many of the proteins that we identified as specific
binders of unmethylated RNA are found in stress
granules, cytoplasmic foci that protect mRNA dur-
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D.; Fu, Y.; Parisien, M.; Dai, Q.; Jia, G.; Ren, B.; Pan, T.; He, C.
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the absence of m A may promote RNA stability or
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Xiao, W.; Adhikari, S.; Dahal, U.; Chen, Y. S.; Hao, Y.
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Cell 2016, 61 (4), 507-19.
regulate the incorporation of mRNA into stress
granules.
Finally, our findings demonstrate that diazirine
nucleotides can mediate higher efficiency photo-
crosslinking than sulfur- or halogen-containing
nucleotides and show that diazirines warrant fur-
ther consideration as nucleic acid photo-affinity
reagents. In principle, with appropriate selection
and placement of photo-crosslinker, the approach
described herein should be adaptable to study the
interactome of any post-transcriptional RNA modi-
fication and provides a powerful tool for decoding
the function of the RNA epitranscriptome.
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Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.;
Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M., Jr.;
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Zhu, T.; Roundtree, I. A.; Wang, P.; Wang, X.; Wang,
Supporting Information. Supplementary Methods,
RNA probe characterization, Supplementary Figures
and proteomics data is available free of charge on the
ACS Publications website.
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AUTHOR INFORMATION
J.; Liu, C.; He, C. Cell Res 2017, 27 (3), 315-328.
7. Zheng, G.; Dahl, J. A.; Niu, Y.; Fedorcsak, P.; Huang,
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Corresponding Author
C. M.; Li, C. J.; Vagbo, C. B.; Shi, Y.; Wang, W. L.; Song, S. H.;
Lu, Z.; Bosmans, R. P.; Dai, Q.; Hao, Y. J.; Yang, X.; Zhao, W.
M.; Tong, W. M.; Wang, X. J.; Bogdan, F.; Furu, K.; Fu, Y.; Jia,
G.; Zhao, X.; Liu, J.; Krokan, H. E.; Klungland, A.; Yang, Y. G.;
He, C. Mol Cell 2013, 49 (1), 18-29.
rkleiner@princeton.edu
Funding Sources
No competing financial interests have been declared.
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Jia, G.; Fu, Y.; Zhao, X.; Dai, Q.; Zheng, G.; Yang, Y.;
ACKNOWLEDGMENT
Yi, C.; Lindahl, T.; Pan, T.; Yang, Y. G.; He, C. Nat Chem Biol
2011, 7 (12), 885-7.
We thank Tharan Srikumar and the Princeton Univer-
sity Mass Spectrometry and Proteomics Core Facility
for proteomics analysis. We thank Jared M. Shulkin
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Kedersha, N.; Ivanov, P.; Anderson, P. Trends
M.; Dewell, S.; Hafner, M.; Williams, Z.; Ohler, U.; Tuschl, T.
Nature 2012, 492 (7429), 382-6.
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Fukuda, T.; Naiki, T.; Saito, M.; Irie, K. Genes Cells
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Chen, E.; Sharma, M. R.; Shi, X.; Agrawal, R. K.;
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Bratic, A.; Park, C. B.; Camara, Y.; Milenkovic, D.; Zickermann,
V.; Wibom, R.; Hultenby, K.; Erdjument-Bromage, H.; Tempst,
P.; Brandt, U.; Stewart, J. B.; Gustafsson, C. M.; Larsson, N. G.
EMBO J 2012, 31 (2), 443-56.
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Kedersha, N.; Panas, M. D.; Achorn, C. A.; Lyons, S.;
Ruzzenente, B.; Metodiev, M. D.; Wredenberg, A.;
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N
a
b
6
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“
m A” probe
N
6
probe 1: GG-m A-C-5-DzU-GUAC-biotin
5
-DzU
O
4-SU
S
5-IU
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
CH3
O
N
O
O
NH
HN
I
O
S
NH
NH
NH
N
N
HN
N
H
N
N
N
N
O
N
O
N
O
HN
NH
O
N
probe 1
.04
probe 3
1 0.04
probe 4
0
1 0.04
1 µM
YTHDC1-
YTH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
“
A” probe
WB: streptavidin
probe 2: GGAC-5-DzU-GUAC-biotin
100
O
N
O
NH2
probe 1
probe 3
probe 4
O
S
N
N
HN
N
H
80
N
N
N
HN
NH
O
N
60
40
20
0
**
6
*
*
**
probe 3: GG-m A-C-4-SU-GUAC-biotin
*
*
*
**
6
probe 4: GG-m A-C-5-IU-GUAC-biotin
0.037
0.11
0.33
1
[
RNA probe] (µM)
GGACU
c
d
6
probe 1 (GGm ACU) probe 2 (GGACU)
6
GGm ACU competitor
competitor
0.001
1 0.001
1 µM
0.005
YTHDC1-
YTH
30
3.3 10 30 µM
YTHDC1-
YTH
WB: streptavidin
WB: streptavidin
120
00
80
120
B probe 1: EC = 46 +/- 14 nM
J probe 2: EC > 1 µM
50
IC = 0.54 +/- 0.24 µM
50
1
B
B
100
50
B
B
B
B
80
60
40
B
6
0
B
40
B
B
J
2
0
20
B
B
J
J
B
B
J
B
J
ACS Paragon Plus Environment0
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B
0.001
0.01
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10
100
0.001
0.01
0.1
1
6
[probe] (µM)
[GGm ACU competitor] (µM)

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GGm ACU binders
GGACU binders
GGACU binders
GGm ACU binders
4
4
3
.5
.0
.5
1
2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
LUC7L3
CAPRIN1
RBM42
YTHDF3 YTHDF1
G3BP2 G3BP1
LRPPRC YTHDC1
YTHDF2
USP10
probe 1
GGm ACU)
probe 2
(GGACU)
6
hυ
hυ
(
SNRPB
CH3
HN
N
NH2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
3.0
2.5
N
N
N
N
N
O
S
O
S
N
HN
NH
HN
NH
N
MEX3B
2.0
1.5
1.0
streptavidin
pulldown
streptavidin
pulldown
FMR1
SAMHD1
ALKBH5
p < 0.05
RNase
elution
RNase
elution
LC-MS/MS
LC-MS/MS
0.5
0
ACS Paragon P-l6us Environment-4
-2
0
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4
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m/z
m/z
log (m A/A spectral counts)
2

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b
Journal of the American Chemical Society
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LRPPRC
FMR1
Agenet
KH1 KH2
RGG
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
582
100
PPR repeats
1
1394
6
B
J
J m A competitor
B
J
IC = 3.3 +/- 1.2 µM
50
6
6
GGA/m ACU
motif
GUA/m ACU
motif
80
B
J
B A competitor
*
B
J
IC = 6.8 +/- 2.5 µM
50
6
0
0
probe:
2
1
8
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
B
J
WB: FLAG
WB: FLAG
B
J
20
0
c
1
1
1
2
2
B
USP10
UCH
J
1
1
798
0
.1
1
10
100
[competitor] (µM)
CAPRIN1
HR1 E-Rich
USP10
HR2
2
2
2
2
2
2
2
2
3
3
3
3
694
d
f
CAPRIN1
RBM42
RRM
480
probe:
2
1
2
1
1
probe:
2
1
**
*
WB: FLAG
WB: FLAG
RRM
*
WB: RBM42
e
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
G3BP1
NTF2
1
E-rich
466
probe 2
probe 6
probe 8
probe 2
probe 1
G3BP1
0.004
3
0.11
3 µM
G3BP1
WB: streptavidin
probe 2 (GGACU)
probe 6 (AAACU)
probe 8 (GUACU)
WB: streptavidin
12
100
B probe 2 (GGACU)
B
EC = 240 +/- 42 nM
5
0
10
8
B
80
60
40
20
6
J probe 1 (GGm ACU)
EC > 3 µM
50
B
6
J
4
*
*
B
J
2
*
B
J
*
*
J
**
B
**
0
0
B
ACS Paragon Plus Environment
0
.001
0.01
0.1
1
10
0.11
0.33
1
[
probe] (µM)
[probe] (µM)