Identification of a signature motif for the eIF4a3–SECIS interaction

eIF4a3, a DEAD-box protein family member, is a component of the exon junction complex which assembles on spliced mRNAs. The protein also acts as a transcript-selective translational repressor of selenoprotein synthesis during selenium deficiency. Selenocysteine (Sec) incorporation into selenoproteins requires a Sec Insertion Sequence (SECIS) element in the 3′ untranslated region. During selenium deficiency, eIF4a3 binds SECIS elements from non-essential selenoproteins, preventing Sec insertion. We identified a molecular signature for the eIF4a3-SECIS interaction using RNA gel shifts, surface plasmon resonance and enzymatic foot printing. Our results support a two-site interaction model, where eIF4a3 binds the internal and apical loops of the SECIS. Additionally, the stability of the complex requires uridine in the SECIS core. In terms of protein requirements, the two globular domains of eIF4a3, which are connected by a linker, are both critical for SECIS binding. Compared to full-length eIF4a3, the two domains in trans bind with a lower association rate but notably, the uridine is no longer important for complex stability. These results provide insight into how eIF4a3 discriminates among SECIS elements and represses translation

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

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

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

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

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

<div><p>Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3’ untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.</p></div

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