Fig 1: Immunofluorescence of endogenous SelS after siRNA treatment in HepG2 cells.HepG2 cells were treated with individual siRNAs as indicated. After 72 hours the cells were fixed and processed for immunofluorescence as described in Materials and Methods. Panel a: untreated cells, b: non-targeting control siRNA, c&d: siRNAs directed at the coding region that target both mRNAs, e: variant 1-specific siRNA, f: variant 2-specific siRNA.
Fig 2: The predicted SL1 structure is conserved. A, The structure annotated alignment derived from the first 50 nucleotides from each SelS 3'UTR using the RNAalifold program. The color code indicates the number of base pair types found at each position: ochre-2, green-3, turquoise-4, blue-5, violet-6. Less saturated colors indicate that this base pair cannot be formed in some of the sequence set. B, Consensus secondary structure prediction of SL1 from RNAalifold. Nucleotides that are marked with black circles indicate locations of compensatory mutations within the sequence set. The probability of a base pair interaction is indicated on a sliding scale from 0 (blue) to 1 (red), as indicated by the legend.
Fig 3: Validation of the SelS-V5 surrogate assay. a. Schematic representation of the SelS-V5/UGA construct. The V5-epitope tag was placed between the UGA-Sec and UAA-stop codons of the human SelS coding region followed by the entire SelS 3' UTR. The UGA-Sec codon was mutated to UAG or UAA as indicated. b. Representative Western blot from three separate experiments of cells transfected with the SelS-V5/UGA and stop codon mutants as well as a vector control. Samples were immunoblotted with a-V5 antibody. The same blot was reprobed for SelS and ß-Tubulin. c. The UGA codon in the SelS-V5/UGA construct was mutated to UGU. d. Western blot of 10 µg of lysate from McArdle 7777 cells transfected with SelS-V5/UGA or decreasing amounts (1 µg, 0.5 µg, 0.25 µg, and 0.125 µg) of lysate from SelS-V5/UGU transfected cells. e. SelS-V5/UGA was transfected into McArdle 7777 cells, immunoprecipitated with a-V5 beads, digested with chymotrypsin, and analysed by LC/MS2. Data represents analysis from three different transfections. The most abundant isotope of the Sec-containing peptide was analysed by MS2 fragmentation. Sequence: RPGRRGPSSGGUG, charge: +3, monoisotopic m/z: 499.2173 Da, [M + H] = 1495.6375. Fragment mass tolerance used for search = 0.6 Da, precursor mass tolerance 10 ppm. Fragments used for search – b; b-NH3 (red), y; y-NH3 (blue). Inset: Isotopic distribution of triply charged Sec-containing peptide.
Fig 4: The region between the seleno-sulfide bond is conserved. A, Sequence alignment of SelS proteins from distantly related species demonstrates that the amino acid sequence between the reported Cys174-Sec188 bond is the most conserved region of the SelS protein. B, Model depicting the carboxy-terminus of SelS showing the potential effect of the truncation of SelS on regulation of the protein function.
Fig 5: The predicted SL 2 structure is conserved. A, The structure annotated alignment derived from the 50 nucleotides immediately downstream of each SelS SECIS element using the RNAalifold program. B, Consensus secondary structure prediction of SL2 generated by RNAalifold. Nucleotides that are marked with black circles indicate locations of compensatory mutations within the sequence set. The probability of a base pair interaction is indicated on a sliding scale from 0 (blue) to 1 (red), as indicated by the legend.
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