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Structural basis of 7SK RNA 5′-γ-phosphate methylation and retention by MePCE

Nature Chemical Biology volume 15, pages 132–140 (2019)Cite this article

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Abstract

Among RNA 5′-cap structures, γ-phosphate monomethylation is unique to a small subset of noncoding RNAs, 7SK and U6 in humans. 7SK is capped by methylphosphate capping enzyme (MePCE), which has a second nonenzymatic role as a core component of the 7SK ribonuclear protein (RNP), an essential regulator of RNA transcription. We report 2.0- and 2.1-Å X-ray crystal structures of the human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. 7SK recognition is achieved by protein contacts to a 5′-hairpin-single-stranded RNA region, thus explaining MePCE’s specificity for 7SK and U6. The structures reveal SAH and product RNA in a near-transition-state geometry. Unexpectedly, binding experiments showed that MePCE has higher affinity for capped versus uncapped 7SK, and kinetic data support a model of slow product release. This work reveals the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of 7SK RNP.

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Fig. 1: MePCE binds and caps 7SK.
Fig. 2: Structural changes in MePCEMT upon binding RNA.
Fig. 3: Sequence- and structure-specific recognition of 7SK by MePCEMT.
Fig. 4: The active site of MePCEMT organizes the cofactor and triphosphate in near transition state.
Fig. 5: MePCEMT multiple-turnover kinetics and binding experiments reveal product and byproduct inhibition and retention.

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Data availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the following accession codes: PDB 6DCB (MePCE–SAH–7SK) and PDB 6DCC (MePCE–SAH–me7SK). All other data generated or analyzed in this study are included in the published article (and its supplementary information files) or are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by NIH grant GM107567 to J.F. and American Cancer Society Postdoctoral Fellowship 126777-PF-14-179-01-DMC to C.D.E. We acknowledge NMR equipment grant NIH S10OD016336 and DOE grant DE-FC0302ER63421 for partial support of NMR and X-ray core facilities. The authors thank M. Capel, K. Rajashankar, N. Sukumar, F. Murphy, I. Kourinov, and J. Schuermann of Northeastern Collaborative Access Team (NE-CAT) beamline ID-24 at the Advanced Photon Source (APS) of Argonne National Laboratory, which are supported by NIH grants P41 RR015301 and P41 GM103403. Use of the APS is supported by DOE under contract DE-AC02-06CH11357. We thank Y. Chen for the help with mass spectrometry data collection and analysis in the UCLA MIC mass spectrometry facility, supported in part by NIH instrumentation grant 1S10OD016387; M. Collazo for help with crystal optimization; and K. Jain, J. Lowenson, and S. Clarke for helpful discussions.

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  1. These authors contributed equally: Yuan Yang, Catherine D. Eichhorn.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Yuan Yang, Catherine D. Eichhorn, Yaqiang Wang, Duilio Cascio & Juli Feigon

  2. UCLA–U.S. Department of Energy (DOE) Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, CA, USA

    Duilio Cascio

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Contributions

Y.Y. and C.D.E. designed and performed experiments, analyzed data, and wrote the paper; Y.W. prepared RNA samples; D.C. helped with X-ray data collection and processing; and J.F. supervised all aspects of the work, analyzed data, and wrote the paper.

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Correspondence to Juli Feigon.

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Yang, Y., Eichhorn, C.D., Wang, Y. et al. Structural basis of 7SK RNA 5′-γ-phosphate methylation and retention by MePCE. Nat Chem Biol 15, 132–140 (2019). https://doi.org/10.1038/s41589-018-0188-z

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