RNA Chemical Proteomics Reveals the N6 ... - ACS Publications

Subscriber access provided by READING UNIV

Communication

An RNA chemical proteomics approach reveals the N6methyladenosine (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 Downloaded from http://pubs.acs.org on November 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

An RNA chemical proteomics approach reveals the N6methyladenosine (m6A)-regulated protein-RNA interactome A. Emilia Arguello, Amanda N. DeLiberto, & Ralph E. Kleiner* Department of Chemistry, Princeton University, Princeton, NJ 08544, USA Supporting Information Placeholder ABSTRACT: 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-crosslinking with synthetic diazirinecontaining RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N6methyladenosine (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.

Chemical modifications on biological macromolecules play a critical role in regulating their function. RNA is extensively modified by cellular enzymes and contains over 100 different post-transcriptional modifications1. While most modifications are found in tRNA, recently, a new class of RNA modifications has been identified. These ‘epitranscriptomic’ modifications2, best exemplified by N6-methyladenosine (m6A)3-4, occur on both internal mRNA sequences 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. The proper regulation of mRNA behavior relies on interactions with a large complement of RNAbinding proteins (RBPs)5 that control RNA splicing,

stability, localization, and translation. Indeed, studies of m6A have demonstrated how recognition of this modification by YTH-domain containing proteins is involved in RNA metabolism6, protein translation7, and splicing8. These findings suggest a general mechanism underlying the function of epitranscriptomic modifications; however, currently we lack a comprehensive understanding of how m6A, as well as other epitranscriptomic modifications, affect the global landscape of cellular protein-RNA interactions. 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 modification. Photo-crosslinking strategies have been widely used to stabilize direct protein-RNA interactions9-10. These approaches rely on the propensity of natural nucleobases or of sulfur- or halogencontaining nucleobase derivatives for UV-induced photochemistry. In contrast, photo-affinity labels such as diazirine11 or benzophenone12, 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. Here, we develop a chemical proteomics approach, relying upon a high-efficiency diazirine-based RNA photo-crosslinker and quantitative proteomics, to profile m6A-regulated RNA-protein interactions. We identify known m6A binders, thereby validating our method. In addition, we find new ‘readers’ of m6A and several proteins that are repelled by this modification. Taken together, our study demonstrates how photo-crosslinking and proteomics can be combined to profile the interactome of modified RNA and ex-

1 ACS Paragon Plus Environment


pands the known repertoire of m6A-regulated protein-RNA interactions. To profile ‘readers’ of m6A using photocrosslinking-based affinity isolation and quantitative proteomics, we needed RNA probes containing: i) m6A, ii) an efficient photo-crosslinker that does not interfere with protein-RNA interactions, and iii) an affinity handle for protein enricha

b

“m6A” probe probe 1: GG-m6A-C-5-DzU-GUAC-biotin HN

CH3

N

O

5-DzU

4-SU

O

S

NH

O

N H

N

N

N

O

N

N N

O

O HN

N

N

HN

NH N

NH

O

0.04

N

N

N

O

N H

100

O

N

N

N

HN

N

probe 3 1 0.04

O

probe 4 1 0.04

1 µM

probe 1 probe 3 probe 4

80

NH

intensity

HN

N

NH

O

WB: streptavidin

O

O

NH2

O

YTHDC1YTH

“A” probe probe 2: GGAC-5-DzU-GUAC-biotin N

5-IU I

NH

probe 1 S

S

60 40

probe 3: GG-m6A-C-4-SU-GUAC-biotin

20

probe 4: GG-m6A-C-5-IU-GUAC-biotin

0

* 0.037

**

**

*

**

*

0.11

**

0.33

1

[RNA probe] (µM)

c

d

probe 1 (GGm6ACU) probe 2 (GGACU) 0.001

1 0.001

1 µM

0.005 YTHDC1YTH

30

120

80

120

B B

B

60 B J 20 B 0 B 0.001

B

J 0.01

x-linking efficiency (%)

B probe 1: EC50 = 46 +/- 14 nM J probe 2: EC50 > 1 µM

40

3.3 10 30 µM

WB: streptavidin

WB: streptavidin

100

GGACU competitor

GGm6ACU competitor

YTHDC1YTH


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100 80

IC50 = 0.54 +/- 0.24 µM

B B

B B

60

B

40

B

20

B

J

B

J

J

0.1

[probe] (µM)

1

0 0.001

0.01

0.1 6

1

10

[GGm ACU competitor] (µM)

B 100

Page 2 of 10

Figure 1. RNA photo-affinity probes for studying m6A-dependent protein-RNA interactions. (a) Structures of RNA probes used in this work. (b) Photocrosslinking 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 differences in cross-linking between probes 1 and 3, and probes 3 and 4 (*: p < 0.05; **: p < 0.005) (c) Photocrosslinking of YTHDC1-YTH with probes 1 or 2. Experiment was performed as in (b) and EC50 values were determined by fitting data to a sigmoidal doseresponse curve. Values represent mean +/- s. d. (probe 1, n = 6; probe 2, n = 3) (see Supplementary Fig. 3 for full WB). (d) Competition of photocrosslinking between YTHDC1-YTH and probe 1 with 5’-GAACCGG-m6A-CUGUCUUA or 5’GAACCGGACUGUCUUA. Experiment was performed 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). ment. Guided by available structural13-14, biochemical6, 13-14, and transcriptomic data3-4, we prepared probe 1 (Fig. 1a), which contains the consensus m6A site in mammalian cells, GGm6ACU. This sequence is sufficient for binding to YTH-domain proteins13-14. We replaced uridine in the GGm6ACU motif with a photo-crosslinkable diazirine-modified uridine residue (5-DzU) and incorporated biotin at the 3’ end of the probe. In addition, we synthesized probe 2 (Fig. 1a), which contains A instead of m6A, but is otherwise identical to probe 1, and probes 3 and 4 (Fig. 1a), which contain m6A flanked by the photo-crosslinker 4-thiouridine (4-SU) or 5-iodouridine (5-IU), respectively. To determine the optimal photo-crosslinker, we assayed photo-crosslinking between m6A-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 photocrosslinking in the 0.037 to 1 µM concentration regime (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 photocrosslinking 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. 1b). A similar trend in probe reactivity was observed


Page 3 of 10

ine-containing probes 1 and 2 effectively recapitulate the affinity and specificity of known m6A readers.

for the YTH domain from YTHDF2 (hereafter ‘YTHDF2-YTH’) (Supplementary Fig. 2). Due to its high photo-crosslinking efficiency, we further characterized probe 1 as well as the corresponding unmethylated probe (2) to confirm that they recapitulated the interactions between YTHdomain proteins and native m6A-modified or unmethylated RNA. We setup photo-crosslinking titrations between probes 1 and 2 and YTHDC1-YTH or YTHDF2-YTH and observed dose-dependent photocrosslinking with probe 1 (YTHDC1-YTH, EC50 = 46 +/- 14 nM; YTHDF2-YTH, EC50 = 0.42 +/- 0.12 µM) (Fig. 1c, Supplementary Fig. 3 and 4) while photocrosslinking with probe 2 proceeded less efficiently (Fig. 1c). We also showed that photo-crosslinking between probe 1 and both YTH-domain proteins can be inhibited by adding an m6A-competitor sequence (YTHDC1-YTH, IC50 = 0.54 +/- 0.24 µM; YTHDF2YTH, IC50 = 4.03 +/- 1.86 µM) (Fig. 1d, Supplementary Fig. 5 and 6), while competition with the identical unmethylated sequence was less efficient, consistent with the reported affinity of these proteins for both GGm6ACU and GGACU RNA sequences13-14. Taken together, our results demonstrate that diazira

We next applied our probes to identify m6A readers in cellular lysate. We set up comparative proteomics experiments in which HeLa cell lysate was photo-crosslinked with 1 µM probe 1 or 2. Crosslinked complexes were then isolated by streptavidin enrichment, eluted from the bead with RNase treatment15, and analyzed by LC-MS/MS to quantify protein abundance (Fig. 2a). We performed 3 independent biological replicates of each proteomics experiment to reliably quantify differences in photocrosslinking between probes 1 and 2, and a ‘volcano plot’ was used to display the results (Fig. 2b, Supplementary Datafile). Our analysis of proteins specifically enriched by probe 1 (Fig. 2b, top right) identified several known ‘readers’ of m6A RNA including YTHDF17, YTHDF26, YTHDF33, 16, and YTHDC18, 13, which showed between 8- to 37-fold preference for the m6A probe (1). We did identify the fifth member of the mammalian YTHdomain protein family, YTHDC2, enriched against probe 1 (but not probe 2) in one

b GGm6ACU binders

GGACU binders

RBM42 4.0

HN N N

probe 2 (GGACU)

hυ

CH3 N

O HN

NH

N

O

N HN

N

S

streptavidin pulldown

NH

S


CAPRIN1

RNase elution

LC-MS/MS

YTHDF2 SNRPB

MEX3B 2.5 2.0 FMR1 p < 0.05

ALKBH5

0.5

intensity m/z

SAMHD1

1.0

LC-MS/MS

YTHDF1

LRPPRC YTHDC1

1.5 RNase elution

YTHDF3

USP10

3.0

NH2

N N

hυ

LUC7L3

G3BP2 G3BP1

3.5

-log10 p-value

probe 1 (GGm6ACU)

GGm6ACU binders

GGACU binders 4.5

intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 -6

-4

-2

0

2

4

6

log2 (m6A/A spectral counts)

m/z

6

Figure 2. Proteomic profiling of the m A interactome. (a) Schematic for comparative proteomics workflow. (b) Vol6 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), although the lack of reproducibility in this data precluded reliable quantification of binding preference. In addition to YTH proteins, we also found ALKBH5 (Fig. 2b), a known m6A demethylase17, exhibiting 2.4-fold preference for probe 1 over probe 2. Interestingly, we failed to identify FTO18, the other major m6A demethylase, suggesting that

this protein may bind less tightly or is less abundant in HeLa cells. We also identified several novel binders to m6A RNA, including LRPPRC, and fragile X-related proteins FMR119 and FXR2, displaying between 2-3-fold preference for probe 1 (Fig. 2b, Supplementary Datafile). We validated the interaction between



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

LRPPRC and probe 1 by photo-crosslinking with lysate generated from 293T cells expressing FLAGtagged LRPPRC. Gratifyingly we observed preferential crosslinking of LRPPRC to the m6A probe (1) over the unmethylated probe (2) (Fig. 3a and Supplementary Fig. 8). In addition, we generated RNA probes containing m6A (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 the m6A-containing probe (7) (Fig. 3a and Supplementary Fig. 8) within this different sequence showing that recognition of m6A does not depend strongly on adjacent nucleotide identity. We also investigated the putative interaction between FMR1 and GGm6ACU RNA. We generated recombinant FMR1 and assayed it for photo-crosslinking to probe 1 in the presence of an m6A-competitor or unmethylated RNA sequence. Using this assay, we observed a significant 2-fold preference in binding of FMR1 to m6A-RNA (IC50 = 3. 3 +/- 1.2 µM) over unmethylated RNA (IC50 = 6.8 +/- 2.5 µM) (Figure 3b, Supplementary Fig. 9 and 10). Unexpectedly, we also identified proteins enriched by the unmethylated probe (2) (Fig. 2b, top left). Several of these proteins are found in stress granules20, including G3BP1, G3BP2, RBM4221, USP10, and CAPRIN1, all exhibiting between to 2.2to 25-fold preference for probe 2. G3BP1 has been reported to interact with USP10 and CAPRIN122, 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 expressing a FLAG-tagged transgene. Our results show that USP10, CAPRIN1, and RBM42 all interact preferentially with probe 2 (Fig. 3c and 3d, Supplementary Fig. 11, 12, 13, 14, and 15), as predicted by our mass spectrometry data. For G3BP1, we generated recombinant protein and performed photo-crosslinking titrations with probes 1 and 2. We measured an EC50 of 240 +/- 42 nM for the reaction between the GGACU probe (2) and G3BP1 (Fig. 3e and Supplementary Fig. 14). In contrast, photo-crosslinking between G3BP1 and the m6A probe (1) proceeded much less efficiently (EC50 > 3 µM), consistent with our mass spectrometry data. We also assayed photo-crosslinking between G3BP1 and related unmethylated probes containing the sequence AAACU (6) or GUACU (8) and found that the GGACU motif was preferred by

Page 4 of 10

4-10-fold over AAACU and 60-100-fold over GUACU (Fig. 3f and Supplementary Fig. 15). Here, we have developed a chemical proteomics approach to profile cellular proteins that bind to m6A-

Figure 3. Characterization of novel protein-RNA interactions. (a) Photo-crosslinking of LRPPRC to probes 1, 2, 7, and 8. Lysate generated from 293T cells transfected with 3x-FLAG-LRPPRC was photocrosslinked 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 photocrosslinking between FMR1 and probe 1. Reactions were performed and analyzed as in Fig. 1d but with 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; *: p < 0.05) . (d) Photo-crosslinking of RBM42 to 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 ) Photocrosslinking of recombinant G3BP1 with unmethylated probes 2, 6, and 8. Experiment was performed and


Page 5 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analyzed as in Fig. 1b (see Supplementary Fig. 15 for 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).

modified RNA. We identified YTH-domain proteins and ALKBH5, known ‘readers’ and ‘erasers’ of this mark, thereby validating our method. In addition, we found novel m6A binders LRPPRC and FMR1 and showed that G3BP1/2, RBM42, USP10 and CAPRIN1 are repelled by the m6A modification. Our results demonstrate that the m6A-regulated protein-RNA interactome is more diverse than previously appreciated. We speculate that these novel protein-RNA interactions may regulate aspects of mRNA metabolism or protein translation, analogous to the known functions of YTH domaincontaining proteins. Indeed, both FMR123-24 and LRPPRC25 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 during periods of stress20. Our findings suggest that the absence of m6A may promote RNA stability or regulate the incorporation of mRNA into stress granules. Finally, our findings demonstrate that diazirine nucleotides can mediate higher efficiency photocrosslinking than sulfur- or halogen-containing nucleotides and show that diazirines warrant further 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 modification and provides a powerful tool for decoding the function of the RNA epitranscriptome. ASSOCIATED CONTENT Supporting Information. Supplementary Methods, RNA probe characterization, Supplementary Figures and proteomics data is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

[email protected] Funding Sources

No competing financial interests have been declared.

ACKNOWLEDGMENT We thank Tharan Srikumar and the Princeton University Mass Spectrometry and Proteomics Core Facility for proteomics analysis. We thank Jared M. Shulkin

for molecular biology assistance. R.E.K. is a Dale F. Frey Breakthrough Scientist of the Damon Runyon Cancer Research Foundation (DFS #21-16) and a Sidney Kimmel Foundation Scholar. All authors thank Princeton University for financial support.

REFERENCES 1. Cantara, W. A.; Crain, P. F.; Rozenski, J.; McCloskey, J. A.; Harris, K. A.; Zhang, X.; Vendeix, F. A.; Fabris, D.; Agris, P. F. Nucleic Acids Res 2011, 39 (Database issue), D195-201. 2. Li, X.; Xiong, X.; Yi, C. Nat Methods 2016, 14 (1), 23-31. 3. Dominissini, D.; Moshitch-Moshkovitz, S.; Schwartz, S.; Salmon-Divon, M.; Ungar, L.; Osenberg, S.; Cesarkas, K.; Jacob-Hirsch, J.; Amariglio, N.; Kupiec, M.; Sorek, R.; Rechavi, G. Nature 2012, 485 (7397), 201-6. 4. Meyer, K. D.; Saletore, Y.; Zumbo, P.; Elemento, O.; Mason, C. E.; Jaffrey, S. R. Cell 2012, 149 (7), 1635-46. 5. Gerstberger, S.; Hafner, M.; Tuschl, T. Nat Rev Genet 2014, 15 (12), 829-45. 6. Wang, X.; Lu, Z.; Gomez, A.; Hon, G. C.; Yue, Y.; Han, D.; Fu, Y.; Parisien, M.; Dai, Q.; Jia, G.; Ren, B.; Pan, T.; He, C. Nature 2014, 505 (7481), 117-20. 7. Wang, X.; Zhao, B. S.; Roundtree, I. A.; Lu, Z.; Han, D.; Ma, H.; Weng, X.; Chen, K.; Shi, H.; He, C. Cell 2015, 161 (6), 1388-99. 8. Xiao, W.; Adhikari, S.; Dahal, U.; Chen, Y. S.; Hao, Y. J.; Sun, B. F.; Sun, H. Y.; Li, A.; Ping, X. L.; Lai, W. Y.; Wang, X.; Ma, H. L.; Huang, C. M.; Yang, Y.; Huang, N.; Jiang, G. B.; Wang, H. L.; Zhou, Q.; Wang, X. J.; Zhao, Y. L.; Yang, Y. G. Mol Cell 2016, 61 (4), 507-19. 9. Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M., Jr.; Jungkamp, A. C.; Munschauer, M.; Ulrich, A.; Wardle, G. S.; Dewell, S.; Zavolan, M.; Tuschl, T. Cell 2010, 141 (1), 129-41. 10. Licatalosi, D. D.; Mele, A.; Fak, J. J.; Ule, J.; Kayikci, M.; Chi, S. W.; Clark, T. A.; Schweitzer, A. C.; Blume, J. E.; Wang, X.; Darnell, J. C.; Darnell, R. B. Nature 2008, 456 (7221), 464-9. 11. Dubinsky, L.; Krom, B. P.; Meijler, M. M. Bioorg Med Chem 2012, 20 (2), 554-70. 12. Kauer, J. C.; Erickson-Viitanen, S.; Wolfe, H. R., Jr.; DeGrado, W. F. J Biol Chem 1986, 261 (23), 10695-700. 13. Xu, C.; Wang, X.; Liu, K.; Roundtree, I. A.; Tempel, W.; Li, Y.; Lu, Z.; He, C.; Min, J. Nat Chem Biol 2014, 10 (11), 927-9. 14. Zhu, T.; Roundtree, I. A.; Wang, P.; Wang, X.; Wang, L.; Sun, C.; Tian, Y.; Li, J.; He, C.; Xu, Y. Cell Res 2014, 24 (12), 1493-6. 15. Michlewski, G.; Caceres, J. F. RNA 2010, 16 (8), 16738. 16. Shi, H.; Wang, X.; Lu, Z.; Zhao, B. S.; Ma, H.; Hsu, P. J.; Liu, C.; He, C. Cell Res 2017, 27 (3), 315-328. 17. Zheng, G.; Dahl, J. A.; Niu, Y.; Fedorcsak, P.; Huang, 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. 18. Jia, G.; Fu, Y.; Zhao, X.; Dai, Q.; Zheng, G.; Yang, Y.; Yi, C.; Lindahl, T.; Pan, T.; Yang, Y. G.; He, C. Nat Chem Biol 2011, 7 (12), 885-7. 19. Jin, P.; Warren, S. T. Hum Mol Genet 2000, 9 (6), 9018.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20. Kedersha, N.; Ivanov, P.; Anderson, P. Trends Biochem Sci 2013, 38 (10), 494-506. 21. Fukuda, T.; Naiki, T.; Saito, M.; Irie, K. Genes Cells 2009, 14 (2), 113-28. 22. Kedersha, N.; Panas, M. D.; Achorn, C. A.; Lyons, S.; Tisdale, S.; Hickman, T.; Thomas, M.; Lieberman, J.; McInerney, G. M.; Ivanov, P.; Anderson, P. J Cell Biol 2016, 212 (7), 845-60. 23. Ascano, M., Jr.; Mukherjee, N.; Bandaru, P.; Miller, J. B.; Nusbaum, J. D.; Corcoran, D. L.; Langlois, C.; Munschauer,

Page 6 of 10

M.; Dewell, S.; Hafner, M.; Williams, Z.; Ohler, U.; Tuschl, T. Nature 2012, 492 (7429), 382-6. 24. Chen, E.; Sharma, M. R.; Shi, X.; Agrawal, R. K.; Joseph, S. Mol Cell 2014, 54 (3), 407-417. 25. Ruzzenente, B.; Metodiev, M. D.; Wredenberg, A.; 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.


Page 7 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Artwork

ACS Paragon Plus Environment

7

a

HN N

CH3

N H

N

O

N

O

O HN

N

N

N

O

N

N

HN

N

N

YTHDC1YTH

N H

N

O N

N

HN

probe 4: GG-m A-C-5-IU-GUAC-biotin

0

100

1 0.001

1 µM

1 0.04

0.037

80

B

60 B J

20 0 B 0.001

B B

J 0.01

1 0.04

0.11

*

1

[RNA probe] (µM)

GGACU competitor

GGm ACU competitor

100 80

30

3.3 10 30 µM

WB: streptavidin

IC50 = 0.54 +/- 0.24 µM

B B

B

B

60

B

40

B

20

J ACS Paragon Plus Environment0 0.001 0.1 1

[probe] (µM)

**

0.33

B

J

J

1 µM

**

** **

120

B

40

*

*

0.005 YTHDC1YTH

B

probe 4

probe 1 probe 3 probe 4

WB: streptavidin

B probe 1: EC50 = 46 +/- 14 nM J probe 2: EC50 > 1 µM

probe 3

O

6


120

0.001

N

O

WB: streptavidin

d

probe 1 (GGm6ACU) probe 2 (GGACU)

NH

40 20

YTHDC1YTH

N

O

I

60

probe 3: GG-m6A-C-4-SU-GUAC-biotin 6

5-IU

NH

O

0.04

80

NH S

c

S

N

100

O

HN O

O

probe 1

O

N

4-SU

NH

intensity

N

5-DzU NH

“A” probe probe 2: GGAC-5-DzU-GUAC-biotin NH2

NH

Page 8 of 10

O

S


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

b

Journal of the American Chemical Society “m6A” probe N probe 1: GG-m6A-C-5-DzU-GUAC-biotin

0.01

0.1

1

B 10

[GGm ACU competitor] (µM) 6

B 100

b


GGACU binders

4.5 4.0

hυ

HN N N

CH3 N

O HN

NH

N

HN

S

streptavidin pulldown RNase elution LC-MS/MS

GGm6ACU binders LUC7L3

CAPRIN1

G3BP2 G3BP1

2.5

YTHDF3 LRPPRC YTHDC1

USP10

YTHDF2

MEX3B

2.0

FMR1 SAMHD1

p < 0.05

ALKBH5

1.0

LC-MS/MS

YTHDF1

SNRPB

1.5

RNase elution

m/z

NH S


RBM42

3.0

O

N N

GGACU binders

3.5

NH2

N N

hυ

probe 2 (GGACU)

-log10 p-value

probe 1 (GGm6ACU)

intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

GGm ACU binders 6

intensity

a

Page 9 of 10

0.5 0 -6 Environment-4 ACS Paragon Plus

m/z

-2

0

2

log2 (m6A/A spectral counts)

4

6

a

b


LRPPRC

100

1394

x-linking efficiency

1 2 PPR repeats 3 1 4 5 GGA/m6ACU GUA/m6ACU 6 7 motif motif 8 9 probe: 2 1 8 7 10 11 12 13 WB: FLAG WB: FLAG 14 15 16 17 UCH 18 USP10 19 1 20 21 HR1 E-Rich HR2 CAPRIN1 22 23 1 24 25 USP10 CAPRIN1 26 probe: 2 1 2 1 27 28 29 30 31 WB: FLAG WB: FLAG 32 33 34 E-rich RRM NTF2 35 G3BP1 36 1 466 37 38 probe 2 probe 39 40 0.004 3 0.11 41 42 G3BP1 43 44 WB: streptavidin 45 46 100 B probe 2 (GGACU) 47 48 EC50 = 240 +/- 42 nM 49 B 80 J probe 1 (GGm6ACU) 50 51 EC50 > 3 µM 52 60 53 B 54 55 56 40 57 58 B 59 20 J 60

c

**

FMR1

798 694

1

B J

80

Agenet

B J

60

B

B 0.01

J

0.1

J

[probe] (µM)

1

IC50 = 3.3 +/- 1.2 µM

B

B A competitor

IC50 = 6.8 +/- 2.5 µM

B

B J

B

20

J

RBM42

1

10

1

probe:

2

RRM 480

1

f

probe 2

*

probe 6

probe 8

G3BP1

3 µM

WB: streptavidin 12 B

10

J

probe 2 (GGACU) probe 6 (AAACU) probe 8 (GUACU)

8 6 4 2

ACS Paragon Plus Environment

10

0

* *

0.11

* 0.33

** **

[probe] (µM)

B J 100

[competitor] (µM)

WB: RBM42

intensity

x-linking efficiency

0 0.001

J m6A competitor

40

*

1

582

J

e

B

RGG

KH1 KH2

J

0 0.1

d

Page 10 of 10

** 1

*

RNA Chemical Proteomics Reveals the N6 ... - ACS Publications

Recommend Documents