RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro
- Greehey Children’s Cancer Research Institute, University of Texas Health at San Antonio, United States
- Department of Biochemistry and Structural Biology, University of Texas Health at San Antonio, United States
- New England Biolabs, United States
- Division of Hematology and Medical Oncology, Department of Medicine, University of Texas Health at San Antonio, United States
Figures
Figure 1 with 1 supplement
Structural similarity, purification of methyltransferases, and substrate designing.
(a–b) Domain architecture of Mod subunit of EcoP15I, human methyltransferase like-3 (METTL3), and human METTL14 methyltransferases (MTases). All three members belong to the β-class of SAM-dependent …
Figure 1—figure supplement 1
Potential mode of DNA recognition.
(a) Blue ribbons, the MTase core (ModB) of EcoP15I (aa 90–132, 169–261, 385–511 from PDB: 4ZCF) in complex with the target DNA strand (orange sticks). The conserved motifs in class β MTases are …
Figure 2
RNA-mediated restriction of methyltransferase like-3 (METTL3)-METTL14 activity.
(a) Methyltransferase activity of METTL3-METTL14 in the presence of various DNA or RNA substrates measured by radiometric assay. CPM, counts per minute. The highest activity was measured with the …
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Figure 2—source data 1
MTase activity on individual RNA/DNA substrates.
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig2-data1-v2.xlsx
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Figure 2—source data 2
Binding isotherms of METTL3-METTL14.
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig2-data2-v2.xlsx
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Figure 2—source data 3
Comparison of MTase activity on different RNA/DNA substrates.
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig2-data3-v2.xlsx
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Figure 2—source data 4
Dose-dependent inhibition of MTase activity by RNA.
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig2-data4-v2.xlsx
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Figure 2—source data 5
RNA-mediated attenuation of MTase activity by intact mass analysis.
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig2-data5-v2.xlsx
Figure 3
Role of RGG motifs and model of RNA-mediated regulation of methyltransferase activity.
(a) Domain architecture of methyltransferase like-3 (METTL3) and METTL14. LH, leader helix; NLS, nuclear localization signal; ZnF1/2, zinc-finger domain 1/2; RGG, arginine-glycine rich repeats …
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Figure 3—source data 1
Binding isotherms of METTL3-METTL14 (-RGG).
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig3-data1-v2.xlsx
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Figure 3—source data 2
Relative MTase activity of METTL3-METTL14 and METL3-METL14 (-RGG).
- https://cdn.elifesciences.org/articles/67150/elife-67150-fig3-data2-v2.xlsx
Tables
Table 1
Equilibrium binding constants.
| Nucleic acid substrate | kd in nM (range of Kd in nM) | |
|---|---|---|
| Full-length METTL3-METTL14 | METTL3-METTL14-RGG | |
| RNA | ||
| rNEAT2 | 2.1 (1.8–2.3) | 21.2 (19.2–23.5) |
| rNEAT2* | 13.0 (12.3–13.8) | 274.1 (150.7–497.2) |
| rTCE23 | 25.6 (23.2–28.5) | 413 (341–502) |
| rPal-top+ bot | 127 (97.2–167) | 257 (132–491) |
| rFoxM1-p1 | 294 (245–354) | 920 (480–1940) |
| r6T | 114 (103–127) | 704 (323–1668) |
| DNA | ||
| d6T | 370 (343–401) | 2097 (1494 – >2000) |
| d6T* | 509 (470–553) | >2000 |
