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Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes

Nature Structural & Molecular Biology volume 12, pages 408–416 (2005)Cite this article

Abstract

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3′ untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G·A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNASec to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G·A pairs, decreases the SBP2–SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.

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Figure 1: Characterization of the 14.5-kDa SECIS-binding protein.
Figure 2: Purification and analysis of L30.
Figure 3: Interaction of L30 with selenoprotein mRNAs in vivo.
Figure 4: Effect of L30 overexpression on UGA-selenocysteine recoding activity.
Figure 5: SECIS-binding activities of L30 and SBP2 in vitro.
Figure 6: Displacement of preformed L30–SECIS and SBP2–SECIS complexes.
Figure 7: Models for UGA recoding.

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Acknowledgements

We thank B. Willard and M. Kinter of the Mass Spectrometry Facilities of the Cleveland Clinic Foundation for performing the mass spectrometry experiments. The liquid chromatography–mass spectrometry instrument used in this study was purchased with funding from the US National Institutes of Health (RR16794) and the State of Ohio Hayes Investment Trust Fund. We also thank C. Gerber for the preparation of rat tissue extracts, P. Copeland for providing the luciferase reporter constructs, W. Merrick for providing ribosomes from rabbit reticulocyte lysate, J. Suryadi (Wake Forest University) for expressing and purifying the M. jannaschii L7Ae protein, and C. Gerber and L. Middleton for reading the manuscript. This work was supported by Public Health Service grant HL29582 (D.M.D.), American Cancer Society grant IRG 95-035-09 (B.A.B.), and the Wake Forest University Science Research Fund (B.A.B.).

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Authors and Affiliations

  1. Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, 44195, Ohio, USA

    Laurent Chavatte & Donna M Driscoll

  2. Department of Chemistry, Wake Forest University, Winston-Salem, 27109-7486, North Carolina, USA

    Bernard A Brown II

Authors
  1. Laurent Chavatte
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  2. Bernard A Brown II
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  3. Donna M Driscoll
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Correspondence to Donna M Driscoll.

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Supplementary information

Supplementary Fig. 1

Size fractionation on a Sephacryl S400 column. (PDF 1724 kb)

Supplementary Fig. 2

Determination of apparent Kd values of L30, SBP2 and L7Ae for the SECIS. (PDF 212 kb)

Supplementary Fig. 3

Characterization of SECIS–protein complexes by supershift analyses. (PDF 217 kb)

Supplementary Methods

Cloning and expression of recombinant proteins. (PDF 21 kb)

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Chavatte, L., Brown, B. & Driscoll, D. Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes. Nat Struct Mol Biol 12, 408–416 (2005). https://doi.org/10.1038/nsmb922

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