Alternative Transcripts and 3′UTR Elements Govern the Incorporation of Selenocysteine into Selenoprotein S

<div><p>Selenoprotein S (SelS) is a 189 amino acid trans-membrane protein that plays an important yet undefined role in the unfolded protein response. It has been proposed that SelS may function as a reductase, with the penultimate selenocysteine (Sec¹⁸⁸) residue participating in a selenosulfide bond with cysteine (Cys¹⁷⁴). Cotranslational incorporation of Sec into SelS depends on the recoding of the UGA codon, which requires a Selenocysteine Insertion Sequence (SECIS) element in the 3′UTR of the transcript. Here we identify multiple mechanisms that regulate the expression of SelS. The human SelS gene encodes two transcripts (variants 1 and 2), which differ in their 3′UTR sequences due to an alternative splicing event that removes the SECIS element from the variant 1 transcript. Both transcripts are widely expressed in human cell lines, with the SECIS-containing variant 2 mRNA being more abundant. In vitro experiments demonstrate that the variant 1 3′UTR does not allow readthrough of the UGA/Sec codon. Thus, this transcript would produce a truncated protein that does not contain Sec and cannot make the selenosulfide bond. While the variant 2 3′UTR does support Sec insertion, its activity is weak. Bioinformatic analysis revealed two highly conserved stem-loop structures, one in the proximal part of the variant 2 3′UTR and the other immediately downstream of the SECIS element. The proximal stem-loop promotes Sec insertion in the native context but not when positioned far from the UGA/Sec codon in a heterologous mRNA. In contrast, the 140 nucleotides downstream of the SECIS element inhibit Sec insertion. We also show that endogenous SelS is enriched at perinuclear speckles, in addition to its known localization in the endoplasmic reticulum. Our results suggest the expression of endogenous SelS is more complex than previously appreciated, which has implications for past and future studies on the function of this protein.</p></div

Elements in the 3′UTR inhibit SelS SECIS activity.

<p>Selenocysteine insertion activity of the two variant UTRs in vitro. The luc/UGA²⁵⁸ reporters with the variant 1 UTR, the variant 2 UTR or the SECIS only were in vitro transcribed and then translated in the presence (blue) or absence (white) of recombinant SBP2-CT. Translation products were analyzed in triplicate for luciferase activity. The results represent the mean of three separate experiments and are expressed relative to the activity of the variant 2 3′UTR in the absence of additional SBP2, which was defined as 100 relative luciferase units. The error bars represent one standard deviation. The numbering in the 3′UTRs of the constructs refers to the nucleotide numbers in GenBank sequences NM_203472 (variant 1) and NM_018445 (variant 2 and SECIS only).</p

The predicted SL1 structure is conserved.

<p><b>A</b>, 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>B</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.</p

SelS localization partially overlaps with the Golgi.

<p>Confocal analysis of endogenous SelS and Golgi localization in HepG2 cells. The gallery of images depicts the z-series through a single cell. The z-axis was 9.53 µm long and each step was 0.37 µm. SelS is depicted in green, while golgin p97 is red with areas of colocalization shown in yellow.</p

Sequences in the distal 3′UTR inhibit SECIS activity.

<p>Selenocysteine insertion activity of deletion mutants of the variant 2 3′UTR in vitro. A stem-loop structure indicates the location of the SECIS element. The luc/UGA²⁵⁸ reporter with the entire variant 2 3′UTR (v2) or portions of the 3′UTR corresponding to Start-SECIS, SECIS-end, or the SECIS only were in vitro transcribed and then translated in the presence of recombinant SBP2-CT. Translation products were analyzed in triplicate for luciferase activity. The results represent three separate experiments and are expressed relative to the activity of v2, which was defined as 100 relative luciferase units. The error bars represent one standard deviation.</p

Immunofluorescence of endogenous SelS after siRNA treatment in HepG2 cells.

<p>HepG2 cells were treated with individual siRNAs as indicated. After 72 hours the cells were fixed and processed for immunofluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062102#s2" target="_blank">Materials and Methods</a>. 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.</p

Predicted elements in the 3′UTR of human SelS variant 2 mRNA.

<p>The location of Stem-loop 1 is indicated by the purple box, while Stem-loop 2 is designated with an orange box. The SECIS element is denoted by bold font. The corresponding structural predictions are indicated for each element. The SECIS element was determined using SECISearch 2.19 (<a href="http://genome.unl.edu/SECISearch.html" target="_blank">http://genome.unl.edu/SECISearch.html</a>), while the structures for the two stem loops were predicted using RNAfold (<a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi" target="_blank">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</a>).</p

The predicted SL 2 structure is conserved.

<p><b>A</b>, The structure annotated alignment derived from the 50 nucleotides immediately downstream of each SelS SECIS element using the RNAalifold program. <b>B</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.</p

SL1 promotes Sec insertion when located in proximity to the recoding site.

<p><b>A</b>, Schematic representation of the constructs used in this assay. The V5 epitope tag was inserted between the Sec (U) and the stop codon of the SelS open reading frame to allow detection of Sec insertion. Either the complete 3′UTR (WT) or the 3′UTR with SL1 deleted (ΔSL1) were included in the Sec constructs. A third construct that replaces the Sec (U) with a Cys (C) was included as a positive control for V5 detection in this assay. <b>B</b>&<b>C</b>, The SelS-Cys-V5 and SelS-Sec-V5 (WT and SL1) constructs were in vitro transcribed and translated, and used for immunoprecipitation (IP) against SelS. The IP reaction was resolved by SDS-PAGE and immunoblotted against the V5 epitope tag. The blot for the SelS-Sec-V5 series was stripped and reprobed for SelS. The experiment was repeated five times with similar results and a representative gel is shown.</p

Human SelS is encoded by two variant transcripts.

<p><b>A</b>, Schematic representation of the human SelS protein. The amino acid numbering refers to human SelS. The arrow indicates the location of the Sec residue at position 188. The ER and cytoplasmic domains are as indicated. TM, transmembrane domain. <b>B</b>, Diagram of the splicing events that generate the two variant SelS transcripts. The numbering refers to the nucleotides in the human 3′UTR sequences. The dashed line indicates the location of the 3′UTR splicing event in variant 1. The stem-loop structure indicates the location of the Selenocysteine Insertion Sequence (SECIS) element. <b>C</b>, qRT-PCR results showing the presence of the variant 1 mRNA in all cell types tested. Levels of variant 1 are expressed as a percent of the total SelS transcripts detected in the same sample. Two independent biological samples were assayed in triplicate. Results are displayed as the mean with error bars indicating one standard deviation. <b>D</b>, Representative blot from Western blot analysis of siRNA treated HEK293 cells. Cells were treated with control non-targeting siRNA (con), siRNAs that target both SelS transcripts (total A and B), or siRNAs that specifically target variant 1 (v1) or variant 2 (v2). Untreated cells were also included in the analysis (-). Total protein lysates from these cells were resolved by SDS-PAGE, transferred to PVDF and immunoblotted with a α-SelS antibody. The relative SelS protein levels were quantified and are expressed as a percent of the levels in the control lane. The same blot was reprobed for GAPDH to serve as a loading control.</p