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CRL2 aids elimination of truncated selenoproteins produced by failed UGA/Sec decoding

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Science  03 Jul 2015:
Vol. 349, Issue 6243, pp. 91-95
DOI: 10.1126/science.aab0515
  • Fig. 1 Selenoprotein degradation by CRL2.

    (A) HEK293T GPS reporter cells expressing selenoproteins from the UTR construct were treated or not treated with dominant-negative Cul2 (DNCul2) and then analyzed. (B) GPS assay for cells cultured in serum-free medium supplemented with various concentrations of sodium selenite. (C) GPS assay for cells cultured in serum-free medium with or without sodium selenite supplement and DNCul2 treatment. (D) A schematic representation and nomenclature of each selenoprotein mutant construct. (E) GPS analysis of selenoprotein mutants in (D). FL and truncated selenoproteins are presented using different x-axis scales to avoid off-scaling. (F) Western blot analysis of SEPHS2 or SELV mutants. FL and truncated (Δ) selenoproteins are indicated by arrowheads. Asterisks mark degradation products from FL SELV. GAPDH and tubulin were loading controls.

  • Fig. 2 Failures in Sec incorporation and selenoprotein degradation.

    (A) Protein stability comparison among various forms of selenoproteins by GPS. (B) Western blot analysis of SEPHS2 or SELV mutants. Asterisks indicate degradation products from FL SELV. (C) The stability of SEPHS2 or SELV proteins expressed from the UTR construct was subjected to cycloheximide (CHX)–chase analysis. (D) Cells expressing SEPHS2 or SELV from the UTR construct were cultured in serum-free medium supplied with a graded increase of extracellular sodium selenite, with or without DNCul2 treatment, and analyzed by Western blotting. The percentage of truncated selenoprotein is shown below. (E) Cells expressing SEPHS2 from the UTR construct were treated with short hairpin RNAs (shRNAs) for SBP2 or eEFSec and then analyzed by Western blotting. (F) The protein stability of SEPHS2 in cells treated with shRNAs for SBP2 or eEFSec was analyzed by CHX chase. (G) A schematic representation of GAPDH artificial selenoprotein (AS) transcripts. Cells expressing wild-type GAPDH or AS were analyzed by Western blotting. The introduced in-frame UGA codon terminated GAPDH at amino acid positions 152, 247, and 301. (H) CHX-chase analysis of GAPDH expressed from AS transcripts in (G).

  • Fig. 3 Identification of the determinants for CRL2-mediated selenoprotein degradation.

    (A) Selenoproteins of various lengths were compared for their stability upon DNCul2 treatment. The truncation site relative to Sec is labeled above. Constructs expressing proteins longer than UGA-terminated proteins carried a UGA-to-UGU mutation. Because the stability varied dramatically among proteins, the plots were scale-adjusted for optimal resolution. The GFP/RFP ratios from separate plots cannot be compared directly. (B) By GPS assay, the stabilization of selenoproteins truncated at various locations after DNCul2 treatment was quantified. (C) The sequences near Sec in SELV, SELK, and SELS. The Sec, its N-terminal glycine, and C-terminal residues are labeled in red, blue, and green, respectively. The 12-residue C-terminal tail (CTT) is marked with a box. (D to E) The protein stability of GAPDH or RAN without or with CTT tags at the C terminus was analyzed. CTTV, CTTK, and CTTS, represent the CTT of SELVΔ, SELKΔ, and SELSΔ, respectively. (F) The stability of GAPDH tagged with various lengths of CTTs. (G) The stability of UGA-terminated selenoproteins with mutations in the glycine N-terminal to Sec.

  • Fig. 4 The BC-box proteins contribute to CRL2 recognition of truncated selenoproteins.

    (A) GPS assay for cells carrying UGA-terminated (Δ) or FL selenoproteins infected with viruses expressing various BC-box proteins. (B) GPS assay for cells expressing Δ or FL selenoproteins treated with shRNAs against BC-box proteins. FL and Δ selenoproteins were presented using different x-axis scales. (C) GST pull-down using lysates expressing hemagglutinin (HA)–tagged BC-box proteins and GST or GST-tagged Δ or FL selenoproteins. (D) GPS assay for cells expressing various forms of SELV or SELK infected with viruses expressing VHL, APPBP2, or KLHDC2. The truncation sites relative to Sec are labeled above. (E) GST pull-down using lysates expressing HA-tagged KLHDC2 and GST or various forms of GST-tagged SELK. (F) GST pull-down using lysates expressing GFP-tagged GAPDH-CTT fusion and GST or GST-tagged BC-box proteins. (G) The protein stability of truncated selenoproteins terminated with various CTTs. (H) The stability of UGA-terminated selenoproteins in various cells.

Supplementary Materials

  • CRL2 aids elimination of truncated selenoproteins produced by failed UGA/Sec decoding

    Hsiu-Chuan Lin, Szu-Chi Ho, Yi-Yun Chen, Kay-Hooi Khoo, Pang-Hung Hsu, Hsueh-Chi S. Yen

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S9

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