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. 2001 Oct;7(10):1442–1453.

The selenocysteine incorporation machinery: interactions between the SECIS RNA and the SECIS-binding protein SBP2.

J E Fletcher 1, P R Copeland 1, D M Driscoll 1, A Krol 1
PMCID: PMC1370188  PMID: 11680849

Abstract

The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.

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Selected References

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  1. Atkins J. F., Gesteland R. F. The twenty-first amino acid. Nature. 2000 Sep 28;407(6803):463–465. doi: 10.1038/35035189. [DOI] [PubMed] [Google Scholar]
  2. Berry M. J., Banu L., Chen Y. Y., Mandel S. J., Kieffer J. D., Harney J. W., Larsen P. R. Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature. 1991 Sep 19;353(6341):273–276. doi: 10.1038/353273a0. [DOI] [PubMed] [Google Scholar]
  3. Copeland P. R., Driscoll D. M. Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis. J Biol Chem. 1999 Sep 3;274(36):25447–25454. doi: 10.1074/jbc.274.36.25447. [DOI] [PubMed] [Google Scholar]
  4. Copeland P. R., Fletcher J. E., Carlson B. A., Hatfield D. L., Driscoll D. M. A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J. 2000 Jan 17;19(2):306–314. doi: 10.1093/emboj/19.2.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Copeland P. R., Stepanik V. A., Driscoll D. M. Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2. Mol Cell Biol. 2001 Mar;21(5):1491–1498. doi: 10.1128/MCB.21.5.1491-1498.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fagegaltier D., Hubert N., Carbon P., Krol A. The selenocysteine insertion sequence binding protein SBP is different from the Y-box protein dbpB. Biochimie. 2000 Feb;82(2):117–122. doi: 10.1016/s0300-9084(00)00192-9. [DOI] [PubMed] [Google Scholar]
  7. Fagegaltier D., Hubert N., Yamada K., Mizutani T., Carbon P., Krol A. Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation. EMBO J. 2000 Sep 1;19(17):4796–4805. doi: 10.1093/emboj/19.17.4796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fagegaltier D., Lescure A., Walczak R., Carbon P., Krol A. Structural analysis of new local features in SECIS RNA hairpins. Nucleic Acids Res. 2000 Jul 15;28(14):2679–2689. doi: 10.1093/nar/28.14.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fletcher J. E., Copeland P. R., Driscoll D. M. Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon. RNA. 2000 Nov;6(11):1573–1584. doi: 10.1017/s1355838200000625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujiwara T., Busch K., Gross H. J., Mizutani T. A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF). Biochimie. 1999 Mar;81(3):213–218. doi: 10.1016/s0300-9084(99)80054-6. [DOI] [PubMed] [Google Scholar]
  11. Grundner-Culemann E., Martin G. W., 3rd, Harney J. W., Berry M. J. Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes. RNA. 1999 May;5(5):625–635. doi: 10.1017/s1355838299981542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hubert N., Walczak R., Carbon P., Krol A. A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs. Nucleic Acids Res. 1996 Feb 1;24(3):464–469. doi: 10.1093/nar/24.3.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hüttenhofer A., Noller H. F. Hydroxyl radical cleavage of tRNA in the ribosomal P site. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7851–7855. doi: 10.1073/pnas.89.17.7851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lesoon A., Mehta A., Singh R., Chisolm G. M., Driscoll D. M. An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine. Mol Cell Biol. 1997 Apr;17(4):1977–1985. doi: 10.1128/mcb.17.4.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Low S. C., Grundner-Culemann E., Harney J. W., Berry M. J. SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy. EMBO J. 2000 Dec 15;19(24):6882–6890. doi: 10.1093/emboj/19.24.6882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Martin G. W., 3rd, Harney J. W., Berry M. J. Functionality of mutations at conserved nucleotides in eukaryotic SECIS elements is determined by the identity of a single nonconserved nucleotide. RNA. 1998 Jan;4(1):65–73. [PMC free article] [PubMed] [Google Scholar]
  17. Romby P., Moras D., Bergdoll M., Dumas P., Vlassov V. V., Westhof E., Ebel J. P., Giegé R. Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea. J Mol Biol. 1985 Aug 5;184(3):455–471. doi: 10.1016/0022-2836(85)90294-3. [DOI] [PubMed] [Google Scholar]
  18. Shen Q., McQuilkin P. A., Newburger P. E. RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA. J Biol Chem. 1995 Dec 22;270(51):30448–30452. doi: 10.1074/jbc.270.51.30448. [DOI] [PubMed] [Google Scholar]
  19. Shen Q., Wu R., Leonard J. L., Newburger P. E. Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein. A bifunctional role for DNA-binding protein B. J Biol Chem. 1998 Mar 6;273(10):5443–5446. doi: 10.1074/jbc.273.10.5443. [DOI] [PubMed] [Google Scholar]
  20. Tujebajeva R. M., Copeland P. R., Xu X. M., Carlson B. A., Harney J. W., Driscoll D. M., Hatfield D. L., Berry M. J. Decoding apparatus for eukaryotic selenocysteine insertion. EMBO Rep. 2000 Aug;1(2):158–163. doi: 10.1093/embo-reports/kvd033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Walczak R., Carbon P., Krol A. An essential non-Watson-Crick base pair motif in 3'UTR to mediate selenoprotein translation. RNA. 1998 Jan;4(1):74–84. [PMC free article] [PubMed] [Google Scholar]
  22. Walczak R., Westhof E., Carbon P., Krol A. A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. RNA. 1996 Apr;2(4):367–379. [PMC free article] [PubMed] [Google Scholar]

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