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. Author manuscript; available in PMC: 2019 Oct 12.
Published in final edited form as: Biochim Biophys Acta Gen Subj. 2018 Apr 12;1862(11):2506–2510. doi: 10.1016/j.bbagen.2018.04.011

Molecular mechanism of Selenoprotein P synthesis

Sumangala Shetty 1, Paul R Copeland 1,1
PMCID: PMC6188828  NIHMSID: NIHMS966057  PMID: 29656121

Abstract

Background

Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

Scope of Review

In this review we elaborate on new data and current models that provide insight into how SELENOP is made.

Major Conclusions

SELENOP synthesis requires a specific set of factors and conditions.

General Significance

As the key protein required for proper selenium distribution, SELENOP stands out as a lynchpin selenoprotein that is essential for male fertility, proper neurologic function and selenium metabolism.

Keywords: selenium, selenocysteine, selenoprotein P, SECIS, SBP2, SECISBP2L

1. Introduction

Identification of the codon for the 21st amino acid, selenocysteine (Sec), led to the first revision of the genetic code in mammals. Sec is the biologically active form of selenium and is co-translationally incorporated into selenoproteins in response to a termination signal or stop codon (UGA). In addition to the classical translation factors, Sec incorporation also requires several other essential factors. These factors include the specialized elongation factor (eEFSec), the selenocysteine tRNA (Sec-tRNASec), a stem loop structure in the 3′ UTR of eukaryotic selenoprotein mRNA known as the selenocysteine insertion sequence (SECIS) element and the SECIS binding protein 2 (SBP2) [1–3]. Together these factors are sufficient for Sec incorporation in vitro, albeit with lower efficiency [4]. However, the precise interplay among these factors during active translation remains largely unclear. Deletion of Sec-tRNASec gene in mice results in embryonic lethality, confirming the importance of selenoproteins [5].

The human selenoproteome consists of products derived from 25 selenoprotein genes. Most selenoproteins catalyze redox reactions within the cell and thus function as antioxidants. Examples include the glutathione peroxidases, deiodinase and thioredoxin reductases [reviewed in 6]. Among the known selenoproteins, selenoprotein P (SELENOP) is unique because it is the only one that possesses multiple selenocysteine residues (10–34 depending on the species). In addition, the SELENOP mRNA has two SECIS elements in the 3′ UTR as opposed to the single SECIS element found in all other selenoprotein mRNAs [Figure 1A; 7]. SELENOP is an extracellular glycoprotein synthesized in the liver and secreted into the plasma and serves to transport selenium to extra-hepatic tissues [8,9]. In addition, an early termination isoform that contains the SELENOP N-terminus also exhibits antioxidant function [10].

Figure 1.

Figure 1

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A) Schematic representation of rat SELENOP cDNA. Vertical lines represent Sec codons. On the 3′ end is the native SELENOP 3′UTR with SECIS-1 and 2. B) Nucleotide alignment (Clustal W) of SELENOP coding region in vertebrates. Black represents >80% identity, grey represents >60% identity and the <below 60% in white. The SELENOP N-terminus that extends from the start codon up to the second UGA spans three-fourths of the coding region. The C-Terminus is approximately 300 nts and contains Sec2 – Sec10. C) Nucleotide alignment (Clustal W) of SELENOP 3′UTR in mammals. Black represents >80% identity, grey represents >60% identity and <below 60% in white. The 3′UTR has two SECIS elements as shown in red. D) Schematic representation of rat SELENOP SECIS1 and 2. SECIS1 is a form 2 with a smaller apical loop and three helices. SECIS 2 is a form 1 with a larger apical loop. The conserved AAR sequence and core motif are shown in red. The horizontal lines represent the boundaries between the different regions of the SECIS elements.

Deletion of the SELENOP gene in mice alters selenium homeostasis and results in both neuronal degeneration and male infertility [11,12]. Although the biological role, enzymatic function and protein domains of SELENOP have been well characterized [reviewed in 13], the mechanism of synthesis remains largely unclear. This is critical since SELENOP serves as an ideal model to study the processive incorporation of multiple Sec residues. Since single Sec incorporation events have been found to be inefficient, the question arises as to how full-length SELENOP can be efficiently made. Here we focus on reviewing the advances so far in elucidating the mechanism of SELENOP synthesis including current models and future directions.

2. Selenoprotein P mRNA Translation

2.1 Coding Region

One of the very early attempts to analyze the mechanism of multiple Sec incorporation events was by the McCarthy group [14]. In this study, expression of a dual luciferase reporter construct with multiple UGA codons (a total of 3 in this study) resulted in a dramatic reduction of the full length product. Since it was established at that time that translation termination competes with Sec incorporation at any given UGA, it was expected that cumulative termination at successive UGA codons would result in a dramatically reduced yield [14]. Thus a non-processive mechanism was proposed for multiple Sec incorporation. As such, non-processive Sec incorporation in SELENOP would result not only in low yields but would also come with high energy cost to the cell. This non-processive Sec incorporation model poses a conceptual challenge, especially since quantitative analysis of SELENOP from rat plasma provides direct evidence that SELENOP is abundant (26–30 ug/ml in rats), which suggests an efficient selenocysteine incorporation mechanism [15]. While the McCarthy study provided new insight into the role of the codon environment in reprograming UGA from termination to Sec incorporation, it did not address multiple Sec incorporation events in the context of SELENOP sequences. Specifically the pig GPX4 SECIS element was used instead of the SELENOP SECIS elements. Furthermore, in the reporter construct the first UGA was from the GPX4 gene of pig and the 2nd and third UGAs were from native human SELENOP, thus putting the first UGA in a non-SELENOP position and codon context. This is especially relevant since the SELENOP N-terminal coding region sequence is not only highly conserved among all vertebrates but also the first UGA position located within this N-terminus is 100% conserved (Fig 1B). The codon context (up to 12 nucleotides) around the first UGA is 96% conserved in mammals [16].

More than a decade after the McCarthy study, a systematic analysis of the codon context of each SELENOP UGA for readthrough efficiency was analyzed using the SELENOP 3′ UTR [16]. Studies using in vitro translation, mutagenesis and sequence alignments, it provided evidence that readthrough at each SELENOP Sec codon is tightly regulated by codon context but is not inversely proportional to termination codon context. For instance, the first UGA in rat SELENOP (Sec1) has the classically described weakest context for translation termination, i.e a U as the fourth base, and thus it is expected to have the highest readthrough. However, Sec1 has lower readthrough relative to Sec 4–6, each of which have a stronger predicted context for termination with a C at the fourth base. Together, UGA codon context studies confirm that the fourth base is not a reliable predictor of UGA readthrough efficiency and implies a role for either larger or more distant sequence determinants.

Subsequent analysis of a subset of SELENOP UGA codons in their native context inserted between two reporter constructs and containing the SELENOP 3′ UTR demonstrated that multiple Sec incorporation follows a processive mechanism [17]. Once readthrough occurs at the first UGA, Sec incorporation efficiency at downstream UGA codons is increased by 10-fold, thus confirming a processive Sec incorporation mechanism. To test the Sec incorporation efficiency at the first UGA in mammalian cells, another study expressed native zebrafish SELENOP cDNA, GST tagged at the N-terminus [18]. The GST tag on the N-terminus was crucial to allow detection of termination at the first UGA. Although termination at the first UGA has been reported in in vitro studies [19], it had never been analyzed in cells. Labeling of cells expressing GST-SELENOP with 75Se showed two bands, one from early termination at the second UGA and the other at ~50 kDa as expected for full length glycosylated protein. Importantly, anti-GST western blot analysis detected high levels of peptide terminating at the first UGA, which confirms in vitro data that indeed Sec incorporation efficiency at the first UGA is the lowest ~ 5–10% but increases dramatically at subsequent UGA’s. The increase in processivity at downstream UGA codons was further corroborated in rat SELENOP [20]. In the presence of supplemental 75Se, in vitro translation of native full length rat SELENOP gene produces a robust single band around 42 kDa which is the expected molecular weight for unglycoslyated full length protein [20]. Together these results confirm that while Sec incorporation efficiency is low at the first UGA, it increases processively at downstream UGA codons.

To determine the driving force for higher processivity at downstream UGA sites, Fixen et al analyzed the effect of distances between UGA sites. They concluded that the efficiency of processive Sec incorporation corresponds inversely to the distance between two consecutive UGA codons [17]. In rat SELENOP the first UGA is located in the N-terminus and is 612 nts upstream of the 2nd UGA, while Sec codons 2–10 are in the C-terminus and are closely spaced (Fig 1B). Thus if distance is the determinant for Sec incorporation efficiency between UGA sites then translation of rat SELENOP mRNA should produce a termination product at the second UGA, which is around 29 kDa and full length protein ~42 kDa. However, in vitro translation of the SELENOP mRNA detected only a 42kDa band by 75Se labeling and no product at ~29 kDa. Further analysis of the 42 kDa product obtained in vitro, revealed that only 13% of the product was full length [20]. Since most of the UGAs in the C-terminus are spaced very close to each other, it is difficult to separate the termination products based on migration. This suggests that in vitro, termination occurs at UGA sites other than the second UGA. In cells and in animal models, translation of the SELENOP mRNA results in a shortened N-terminal isoform terminating at the second UGA besides termination at the natural stop codon UAA [7,10,18]. However, the ratio of the shortened N-terminal isoform to full length product varies depending upon cell type. For example, unlike kidney cells, liver cells produce several fold more full length protein relative to the shortened N-terminal isoform which suggests that distance may not be the only factor (unpublished results). The discrepancy in the SELENOP termination sites depending upon the expression system used and also cell type, suggests that in cells additional determinants in the SELENOP mRNA may influence the balance between termination and Sec incorporation.

Our analysis of the native SELENOP mRNA found that in vitro processivity was driven by increasing selenium concentrations. Processive but not single Sec incorporation was stimulated >4-fold by supplemental selenium. This led to the proposal of the channeling mechanism for multiple Sec incorporation events [20]. The channeling mechanism suggests that components of the protein synthesis machinery are highly organized on the cytoskeleton so as to allow all the intermediates in the process to interact and channel from one to another without dissociating [21–23]. Although the initial channeling theory, suggested a need for the cellular framework to increase translation efficiency, another study showed that in continuous cell free systems, the tRNAs and the initiation and elongation factors all remain bound to the protein synthesis machinery and are not available as freely diffusible molecules [24]. Thus upon selenium stimulation, the coupling of the increased aminoacylation of Sec-tRNASec with the active Sec incorporation complex might be a crucial factor for processive SELENOP synthesis.

To address the role of the SELENOP coding region sequence, an artificial selenoprotein consisting of the luciferase coding region that was modified to contain 10 Sec codons spaced exactly as found in human SELENOP was synthesized and translated in vitro. This yielded a very weak and diffused product [25]. In contrast, the wild type human SELENOP mRNA yielded a robust ~47 kDa band as expected. This confirms the importance of the SELENOP coding region sequence in determining efficiency. In vitro both the N- and C- terminal native SELENOP sequences are required for optimal expression [25]. A cis-acting RNA sequence called a Sec redefinition element (SRE) was identified in SELENON [26,27]. By analogy, the conserved SELENOP coding sequence may contain a similar SRE. To identify regulatory elements or SREs, bioinformatic analysis of the SELENOP coding region was employed. A comparative search for RNA structures identified four discrete regions in the SELENOP coding region, two of which were located in the N-terminus and two in the C-terminus [25]. The N-terminal structures were termed as initiation stem loop (ISL) and Sec redefinition element (SRE1) while the C-terminal structures were termed as SRE2 and SRE3. Functional analysis of the ISL structure, which overlaps the signal peptide, showed that it affected overall translation initiation rate. On the other hand, mutations in SRE1 which is located downstream of the first UGA, reduced Sec insertion at the first UGA in cultured cells. The role of SRE2 and 3 have yet to be analyzed. Recognition of coding region elements establishes a more complex regulation. In this model, the authors predict that secondary and tertiary interactions within and/or between the coding region SREs and the SELENOP 3′ UTR coordinate recoding at the multiple SELENOP UGA codons and rates of translation initiation and elongation [25]. In addition, our analysis of the SELENOP N-terminal sequences (Shetty and Copeland, unpublished data) using the full length native mRNA, identified a unique sequence important specifically for efficiency of processive Sec incorporation. We also found that deletions in the N-terminal sequences affected secretion as well.

Our current understanding of the SELENOP coding region implies a model in which cis- acting RNA structures and sequences along the entire SELENOP coding region regulate codon redefinition efficiency. Whether these RNA elements interact with cellular factors to alter ribosome conformation for Sec codon recognition or simply stimulate and/or stabilize the already known Sec incorporation machinery needs to be further investigated. What is certain, however, is that the SELENOP coding region sequence is unique and required to efficiently drive multiple Sec incorporation events.

2.2 SELENOP 3′ UTR

Similar to the SELENOP coding region, the SELENOP 3′ UTR is also highly conserved among mammals (Fig 1C). The SELENOP 3′ UTR has two SECIS elements. As shown in Figure 1D, SECIS elements exhibit a conserved K-turn motif in the internal loop made up of GA:GA base pair (shown in red and termed as SECIS core), which is recognized by its cognate binding protein, SBP2 [1,28,29]. In addition, consensus SECIS elements also carry a stretch of conserved adenines (or very rarely cytosines) in the apical loop. SECIS elements are classified into separate categories termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 [30]. SELENOP SECIS-1 is predicted to be of form 2 and SECIS-2 of form 1. However, they both possess the SECIS core motif in the internal loop and the conserved AAR motif in the apical loop. As mentioned before, the number of UGA codons in SELENOP may vary from 10–34 depending on the species. However, irrespective of the number of Sec codons, evolutionarily all SELENOP genes identified so far, contain two SECIS elements. This implies that the two SECIS elements may have distinct regulatory functions.

Stoytcheva et al analyzed for the first time the role of the SELENOP 3′ UTR using the N-terminus GST-tagged zebrafish SELENOP cDNA [18]. Interestingly, analysis of the 3′ UTR mutants in which either of the SECIS elements were deleted revealed that the loss of SECIS-2 had no deleterious effect on processive Sec incorporation while the deletion of SECIS-1 resulted in a complete loss of Sec incorporation downstream of the 2nd UGA [25]. These results were corroborated even in in vitro studies [20]. In native SELENOP mRNA the two SECIS elements are arranged in a S1S2 manner thus positioning SECIS 1 closer to the first UGA than SECIS 2 (S1S2). To analyze if proximity to the first UGA codon played a role, a mutant with SECIS-2 positioned closer to first UGA was tested. Repositioning the SECIS-2 element did not render it processivity. Furthermore duplication of SECIS 2 increased Sec incorporation efficiency at the first UGA but still terminated at the second UGA [18]. Thus, both in in vitro translation and in transfected cells processive Sec incorporation does not require SECIS-2 [18].

These data raise the question of what role SECIS-2 may play in Sec incorporation into SELENOP. Based on the abundance of termination product detected at the first UGA, Stoytcheva et al speculated that the Sec incorporation efficiency at the first UGA by SECIS-2 may be low, although the decoding efficiency of SECIS-2 was not yet analyzed at that time. Thus, the authors proposed that the circularization of the SELENOP mRNA during translation initiation, positions SECIS- 2 at the first UGA for slow decoding, which is then carried forward by SECIS-1 [18]. This then raises the question why do we need slow decoding at the first UGA? The first UGA in the SELENOP gene is the only Sec codon positioned upstream of introns and can potentially invoke the nonsense mediated decay (NMD) pathway. NMD is a quality control mechanism that detects the presence of a ribosome stalling at a premature termination codon (PTC) located >50–55 nucleotides from the exon-exon junction [31]. Thus in the event that an essential component of the Sec incorporation machinery is unavailable, the naturally low efficiency of decoding at the first UGA will either be dramatically reduced or will not occur, thereby resulting in stalling ribosomes and NMD. Thus, the slow decoding at the first UGA functions as a checkpoint to determine if Sec incorporation will occur at the downstream UGAs [18].

In a later study, analysis of decoding efficiencies of SECIS elements to incorporate at the first UGA determined that SECIS-2 activity was indeed six fold lower than SECIS-1 [17]. To analyze if this was true even in mouse models, ribosome profiling was conducted on mouse liver with either SECIS-1 or 2 deleted. Increased ribosome footprint density upstream of the first UGA upon SECIS-2 deletion but not in SECIS-1 deletion was interpreted as slow decoding at the first UGA [25]. In addition, mice with SECIS-2 deletion displayed a dramatic 85% reduction in total plasma SELENOP (15% of wild type) [32]. More recently, analysis of a conserved coding region Sec redefinition element (SRE1), showed that in vitro SRE1 regulation of the first UGA in presence of SECIS-2 alone is dependent on selenium status [25]. Thus together these findings suggest that in transfected cells and in in vitro assays SECIS-2 is dispensable but the distinct role of SECIS-2 cannot be overruled for endogenous SELENOP expression and may include tight post transcriptional regulation.

However, these results raise some very important mechanistic questions. If all SECIS elements have the core motif in the internal loop and the conserved sequence in the apical loop, how do they differ in function? Our lab recently identified a novel determinant in SELENOP SECIS-1 that is required for processive Sec incorporation (presented at the Se2017 conference, manuscript in preparation). A mutation in this sequence resulted in the conversion of SECIS-1 from a processive to a nonprocessive SECIS element, but the mechanism by which this sequence promotes processive Sec incorporation is unknown. Presumably, this sequence is involved in modifying SECIS binding protein function or recruiting novel SECIS binding proteins. Ribosome profiling of wild type mouse liver identified a region of RNAseI protection overlapping SELENOP SECIS-1 in both wild type mice and in SECISBP2 knock out mice, suggesting the presence of a large ribonucleoprotein (RNP) complex [25,33].

Another lingering question is the function for the considerable amount of conserved sequence in the SELENOP 3′ UTR that lies outside of the SECIS elements. More recently, the systematic consecutive deletion analysis of ~150 nt segments in the SELENOP 3′ UTR also confirmed that SECIS-1 was sufficient for processive Sec incorporation [20] and the rest of the UTR was not required. Surprisingly, replacement of the entire 3′ UTR with just the SELENOP SECIS-1 alone or the SECIS from rat GPX4 was sufficient to drive processivity in vitro [20]. It is possible that although in vitro the non-SECIS 3′ UTR sequences appear not important, in tissues they may have yet unidentified roles. Further analysis of the mechanism by which SECIS-1 and the SELENOP 3′ UTR promotes processive Sec incorporation is underway.

Overall it is clear that the complex network of interactions between elements at the two ends of the SELENOP mRNA may serve multiple functions. Currently we know these to include slow decoding at the first UGA by SECIS-2, processive Sec incorporation by SECIS-1, and efficiency of multiple Sec incorporation.

3. Future Directions

While recent studies suggest that the SELENOP mRNA is evolutionarily optimized for efficiency and processivity, several mechanistic questions remain unanswered. This includes the overall structure of SELENOP mRNA, how does SRE1 stimulate Sec incorporation efficiency at the first UGA, the role of SRE 2 and SRE3, the identification and the role of RNP complex on SECIS-1 during multiple Sec incorporation and the RNP landscape during active multiple UGA codon redefinition. In addition, how does Sec incorporation at first stimulate Sec incorporation efficiency downstream? Does the Sec incorporation machinery remain stably complexed with the ribosome or the protein synthesis machinery after incorporation at the first UGA or is channeling a regulator of efficiency at downstream UGA codons? Are the coding region sequences required to recruit and channel the Sec incorporation machinery?

Reconstitution of the Sec incorporation machinery in a Sec naive system namely the wheat germ lysate determined that while known Sec incorporation factors were sufficient for single Sec incorporation it was unable to support recoding at multiple Sec codon, thus suggesting a role for novel cellular factors [20]. Thus besides SBP2 what are the other potential regulators of processive Sec incorporation? Could this include other SECIS binding proteins such as nucleolin, eL30, eIF4A3 or the SBP2 paralog SBP2L? How are the differential termination sites in SELENOP regulated? Further investigation into the mechanism of processive Sec incorporation is important for deeper understanding of the protein synthesis machinery.

To answer all the above mechanistic question future research should include stabilization and isolation of the active complex of processive Sec incorporation for structure function analysis. Also, examination of the RNP complexes in this complex should provide novel insights into the potential mechanism. Future directions should focus on analyzing endogenous SELENOP mRNA and RNP using gene editing techniques. Analysis of this nature will not only decipher processive Sec incorporation but will advance and modify our current understanding of cellular translation.

Highlights (for review).

  1. Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors.

  2. Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

  3. SELENOP synthesis requires a unique set of factors and RNA structures.

  4. SELENOP is essential for male fertility, proper neurologic function and selenium metabolism.

Acknowledgments

Funding: This work was supported by the National Institutes of Health grant numbers R01GM077073 and R21HD083616.

Footnotes

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