Table 1.
Oligonucleotide sequences used in this study.
Fig 1.
Conservation in the SELENOP 3’ UTR.
A) Diagram and multiple sequence alignment of the mammalian SELENOP 3’ UTR. The alignment of sequences from the species indicated was performed using the MUSCLE algorithm, identical positions are black.
Fig 2.
RNA affinity chromatography identifies PTBP1 and other SELENOP 3’ UTR binding proteins.
A) Diagram of the components used for glutathione agarose affinity chromatography and RNA constructs used. B) SDS PAGE analysis of rat liver proteins eluted from a GST-MS2 column bound with either control RNA, SELENOP 3’ UTR (FL 3’ UTR), and the interSECIS deletion mutant (ΔInter). The gel was stained with Coomassie and the bands of interest excised from the gel for LC MS/MS analysis. C) SDS PAGE and D) immunoblot analysis of proteins eluted from RNA affinity experiments as described in A) except that HepG2 lysate was used. The immunoblot was probed with anti-PTBP1 antibody. E) Peptide counts and cognate genes that were identified by LC MS/MS MudPIT analysis after RNA chromatography using control RNA (Cntl), wild-type SELENOP 3’ UTR (WT) and an RNA lacking the first 273 nt of the SELENOP 3’ UTR (Δ273). The ratio of peptide counts from WT UTR versus control RNA is shown (WT:Cntr). The genes shown here correspond to those with a ratio of 5 or above and a peptide count of 10 or above.
Fig 3.
Recombinant PTBP1 binds directly to the SELENOP 3’ UTR.
A) Line diagram of the SELENOP 3’ UTR with canonical U-rich PTBP1 binding sites (UCUU) annotated as green arrows. The four regions that were mutated are indicated with arrows. B) SDS PAGE of recombinant GST-PTBP1 protein UV-crosslinked to 32P-UTP labeled RNA fragments as indicated in A). As a negative control, GST-MS2 coat protein was used as indicated.
Fig 4.
Editing of the SELENOP gene in HepG2 cells.
A) Diagram of the SELENOP 3’ UTR indicating the sequences deleted by CRISPR/Cas9 genome editing. Sequence traces of the deleted regions are shown below. B) genomic PCR (left panel) and RT-PCR (right panel) with primers flanking the deletion sites. C) Lysates from wild-type (WT) and interSECIS deletion (ΔInter) and preSECIS deletion (ΔPre) HepG2 cells were subjected to RNA-IP using the anti-PTBP1 or anti-FLAG control antibodies. RNA was extracted from immunoprecipitates and analyzed for SELENOP mRNA levels by limited-cycle RT-PCR.
Fig 5.
Hydrogen peroxide treatment reveals a regulatory role for the interSECIS sequence.
A) Wild-type (wt) or interSECIS deletion mutant HepG2 cells were treated with the hydrogen peroxide concentrations indicated and 100 nM 75Se-selenite for 6 hours. 30 μl of conditioned medium was analyzed by SDS PAGE followed by immunoblot (top) or phosphorimaging (bottom). Full length (FL) and truncated (term) SELENOP resulting from early termination at the second UGA codon are indicated with arrows. B) Quantitation of the band corresponding to full length (FL) SELENOP from the phosphorimage normalized to quantitation of bands from the stained gel. C) Total RNA was isolated from these cells and analyzed by qRT-PCR normalized to actin and the wt set to 1. For B and C, data were plotted as the mean with error bars showing standard deviation. A Student’s t-test was used to calculate the p values shown on three biological replicates.
Fig 6.
Limiting selenium reveals a role for the PreSECIS region.
A) HepG2 cells were incubated with increasing concentrations of sodium selenite as indicated. 30 μl of conditioned medium was analyzed by SDS PAGE followed by immunoblot probed with anti-SELENOP antibody. B) Same as in A) except 75Se-selenite was used to supplement. SDS-PAGE of conditioned media (top panel) or lysate (bottom panels) was analyzed by phosphorimage analysis and Coomassie Stain (top panel), normalized to quantitation of bands from the stained gel. C) Quantitation of the phosphorimage data derived from conditioned media. D) Quantitation of all radioactive bands in the phosphorimage data derived from lysate. E) Total RNA was isolated from the cell lines indicated with and without 50 nM selenium supplementation and analyzed by qRT-PCR normalized to actin and the wt set to 1. F) A plot of the inferred translational efficiency based on the RNA:protein ratio normalized to wild-type. For all quantification, a Student’s t-test was used to calculate the p values shown on three biological replicates.