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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Biol Trace Elem Res. 2019 Jul 24;192(1):18–25. doi: 10.1007/s12011-019-01827-y

New Directions for Understanding the Codon Redefinition Required for Selenocysteine incorporation

Michael T Howard 1, Paul R Copeland 2
PMCID: PMC6801069  NIHMSID: NIHMS1535642  PMID: 31342342

Abstract

The fact that selenocysteine (Sec) is delivered to the elongating ribosome by a tRNA that recognizes a UGA stop codon makes it unique and a thorn in the side of what was originally thought to be a universal genetic code. The mechanism by which this redefinition occurs has been slowly coming to light over the past 30 years, but key questions remain. This review seeks to highlight the prominent mechanistic questions that will guide the direction of work in the near future. These questions arise from two major aspects of Sec incorporation: 1) novel functions for the Sec insertion sequence (SECIS) that resides in all selenoprotein mRNAs and 2) the myriad of RNA binding proteins, both known and yet to be discovered, that act in concert to modify the translation elongation process to allow Sec incorporation.

Keywords: Selenocysteine, selenium, SECIS, RNA binding proteins

Introduction

The molecular ballet that permits the transformation of what would ordinarily be a translation termination signal into a selenocysteine (Sec) codon is performed by an array of unique factors that momentarily replace the function of canonical translation elongation factors. The players are able to interpret the premature termination codon as a Sec codon and then deliver Sec to the UGA codon. They include the Sec insertion sequence (SECIS) that resides in the 3’ UTR of all selenoprotein mRNAs, the SECIS binding protein, SECISBP2 (SBP2), the Sec-specific translation elongation factor, eEFSec and the unique transfer RNA, Sec-tRNASec. Figure 1 illustrates the basic features of the SECIS (Figure 1A), and the factors shown in Figure 1B are sufficient to promote Sec incorporation [1]. While the mechanism of action and the multitude of factors that work to regulate the process are coming into focus (see Figure 2 below), many fundamental questions remain about how this system works. Below are a series of key unanswered questions that may help to guide the path forward.

Figure 1.

Figure 1.

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The SECIS element is comprised of distinct segments as shown. The residues shown in red are the only universally conserved features of the sequence.

Figure 2.

Figure 2.

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A model for Sec incorporation that incorporates a role of nuclear SecRNP assembly in building the complex that will allow UGA recoding in the cytoplasm. SECISBP2 is designated as SBP2.

I. SECIS elements: RNA structures with multiple functions in selenoprotein synthesis

Why is the SECIS element is so complex?

Messenger RNA plays an active role in regulating the rate and accuracy with which its cognate polypeptide is synthesized. Much of this regulatory functionality lies within the 3’ untranslated region (3’ UTR), which may contain one or more structural signals that typically recruit a myriad of RNA binding proteins. Some of these regulatory mechanisms are general in nature (translational attenuation), while others are quite specific. SECIS elements are paradigmatic examples of these latter structures. However, if SECIS element function was singular, then one would expect a great deal of sequence similarity across all mRNAs that encode selenoproteins. In fact, quite the opposite is true. For a given selenoprotein mRNA, SECIS element sequences are well conserved across species [2], but there is very little conservation among the different selenoprotein mRNAs. In fact, the only two conserved features are the central AUGA:AG sequences, a canonical kink-turn structure [3], and the conserved “AAR” motif in the terminal loop (Figure 1A). This lack of conservation is consistent with the finding that the efficiency of Sec incorporation is vastly different depending on the identity of the SECIS element [2]. While SECISBP2 binding affinity would be the obvious determining variable, there is very little difference in binding affinities across the SECIS elements [2,4]. Recent work has demonstrated unique functions of SECIS elements such as allowing processive Sec incorporation at multiple UGA codons in cis [5] and responding to cellular selenium levels [6]. With these tantalizing new functions, the era during which we determine the mechanisms behind variable SECIS function has just begun.

How does the AAR motif in the SECIS apical loop contribute to Sec incorporation?

Since the initial identification of the eukaryotic SECIS element [7], no function has been assigned to the AAR motif that was identified more than 25 years ago [8]. Mutations in the terminal loop that disrupt the conserved AAR motif completely eliminate the Sec incorporation function but have no effect on SECISBP2 binding, which occurs at the kink-turn motif [9]. Additionally, AAR mutations have no impact on the SECISBP2/SECIS complex to recruit eEFSec [10]. It is tempting to speculate that this part of the SECIS element is making direct contacts with the ribosome, perhaps playing a role in Sec-tRNASec accommodation. However, the fact that some selenoprotein mRNAs harbor SECIS elements with a CCC motif in place of the AAR suggests that the function is not acting on a universal feature like ribosomes, but something that would co-vary with the SECIS element. The most likely candidate is the selenoprotein mRNA itself, perhaps the previously identified Sec recoding element [SRE; 11]. The identification of coding region sequence elements that modulate Sec incorporation provide compelling evidence for this hypothesis, but mechanistic evidence is lacking [11,12,6].

Why don’t SECIS elements work in trans?

In prokaryotes, the SECIS element is adjacent to the UGA codon and is an integral part of the decoding complex [13]. As such, there is no need for the separation of SECIS binding and elongation factor function, and the bacterial version of eEFSec directly binds to the SECIS element [14–16]. Considering the vast distance between the UGA codon and some SECIS elements (up to ~3KB), there is no obvious reason why a separate pre-formed SECISBP2/SECIS complex couldn’t interact with an elongating ribosome and promote Sec incorporation. By definition, of course, the Sec incorporation system cannot allow this because then the SECISBP2/SECIS complex on one mRNA would be able to suppress UGA stop codons on another. This phenomenon may provide indirect evidence for the concept that formation of a selenoprotein mRNA that is competent for Sec incorporation is a temporally restricted process of assembling a complex Sec-specific ribonucleoprotein (“SecRNP”). This idea is more fully developed below, but in the context of SECIS function, one scenario is that the terminal loop is making weak contacts with the mRNA sequence flanking the UGA codon, perhaps at an SRE. This interaction would be facilitated by SECISBP2 and/or other RNA binding proteins that bridge the SECIS and UGA by taking advantage of the lower energy barrier created by the cis relationship.

Why does the SELENOP mRNA have two SECIS elements?

In mammals there is only one selenoprotein, SELENOP, that contains multiple UGA codons, each of which encodes Sec. SELENOP uses two SECIS elements to facilitate decoding of the 10 UGA codons. Two independent studies looking at directed mutation of the two SELENOP SECIS elements, in transfected cells or transgenic mice, have come to the same conclusion: The proximal SECIS element (SECIS 1) is used for processive decoding of the 9 UGA codons near the 3’ end of the CDS, whereas, the distal SECIS (SECIS 2) is only able to decode the first UGA [17,18]. While these studies clearly showed that SECIS 2 was able to incorporate Sec at the first UGA, it did not demonstrate that it is necessary for such. In fact, subsequent work has clearly shown that the first SECIS is sufficient for SELENOP synthesis both in vitro and in transfected cells [19,5,18,20]. In fact, the only detectable effect of eliminating the 2nd SECIS element was a slight increase in full length SELENOP production in vitro or transfected cells [19,18,21]. Although, it should be noted that deletion of SELENOP SECIS1 or SECIS2 in mice has been shown to have distinct effects on SELENOP selenium content and length [20]. While the exact nature of these isoforms was not determined, the in vivo and in vitro data foreshadows later work that implicates SECIS 2 as a regulator that responds to selenium concentrations [6]. This latter proposed function is tantalizing as it may allow for switching SELENOP from bifunctional (selenium transport plus thioredoxin activity) to monofunctional (just thioredoxin activity) under conditions where selenium levels are low, thus permitting the persistence of its antioxidant function even when the full-length protein cannot be made.

II. SecRNPs: RNA ribonucleoprotein complexes involved in selenoprotein synthesis

Eukaryotic cells encode thousands of ribosome-binding proteins (RBPs), each of which has unique RNA-binding activity and characteristic protein-protein interactions. This remarkable diversity of combinations gives rise to a unique ribonucleoprotein (RNP) complex for every mRNA [22,23]. Proper assembly of the RNP is required for pre-mRNA splicing, nucleotide modification, poly-adenylation, nuclear export, RNA localization, translation and turnover. Further, the nature of the RNP is dynamic with binding and assembly initiating immediately after transcription in the nucleus and culminating with recruitment and/or removal of RBPs in the cytoplasm.

Layered on top of the functions shared by all mRNAs, selenoprotein mRNAs must assemble a SecRNP complex that reprograms the ribosome to decode UGA as selenocysteine. At the same time the SecRNP must avoid the mRNA surveillance mechanisms that would normally target an mRNA carrying a premature termination codon for degradation. Through rigorous biochemical and molecular biology studies, a number of RBPs that have specific interactions with the unique SECIS element have been identified [24–27] (Figure 2). Many of these factors have been shown to be capable of shuttling between the nucleus and cytoplasm leading to models proposing that at least some steps of the SecRNP complex formation initiate within the nucleus [28,29].

Resolution of the outstanding questions centered around the unique SECIS features highlighted above will likely require a deeper understanding of how the SecRNP is assembled over the course of selenoprotein mRNA biogenesis in the nucleus, transport to the cytoplasm, and translation. Below we highlight unresolved questions regarding the role of individual proteins and their assembly into a functional SecRNP capable of supporting and regulation selenoprotein translation.

What are the roles of SECISBP2 domains and its interaction with EEFSEC?

SBP2 possesses 3 phylogenetically and biochemically characterized domains: 1) A poorly conserved unstructured N-terminal domain of approximately 400 amino acids; 2) a central “Sec incorporation domain (SID)” that is required for Sec incorporation but not SECIS binding; 3) a C-terminal RNA binding domain that is required for SECIS binding. Interestingly, the first two of these domains are phylogenetically unique, having apparently evolved for the sole purpose of Sec incorporation. The only conserved domain in SECISBP2 that is not unique in biology is the SECIS binding domain, which is a canonical L7Ae RNA binding domain [30,31]. Interestingly, however, this domain in isolation has a very low affinity for SECIS RNA, and the addition of the SID as a separate polypeptide to a binding reaction, increases binding affinity many fold. In fact, these two domains are able to support the full Sec incorporation reaction when present as separate proteins, and they form a stable complex when the SECIS element is present [32]. Besides playing a role in enhancing SECIS binding, the SID also enhances eEFSec recruitment [10]. Since some mutations in the SID eliminate Sec incorporation without affecting SECIS binding or eEFSec recruitment, there remains an as-yet unmeasured activity of SECISBP2 that is essential.

What are the roles of SECISBP2 interactions with the ribosome?

The original purification of SECISBP2 indicated that it elutes from gel-filtration chromatography with a molecular mass of ~500 kDa, suggesting that it exists in a large functional complex [26]. Further studies of SECISBP2 have shown that it can interact with the ribosome through interactions with the ribosomal 28S RNA [31,33]. It is likely that this function is responsible for somehow creating the optimal binding site for the eEFSec ternary complex on the ribosome, but it could also play a role in more downstream activities like GTP hydrolysis or Sec-tRNASec accommodation, or even tRNASec translocation to the E-site. Indeed an analysis of the ribosomal conformational changes that take place upon SECISBP2 binding revealed modifications at E site proximal locations [34]. The focus now must be on generating stable intermediate complexes that can help determine the order of events as well as set the stage for structural analysis. Since there are no known small molecules that inhibit any of the steps in Sec incorporation, we may have to rely on identifying dominant negative acting mutant proteins that will stabilize intermediate steps.

Why is SECISBP2 not always required?

The observations that SECISBP2 interacts directly with EEFSEC lends itself to a model whereby SECISBP2 is the central player that tethers the EEFSEC ternary complex to selenoprotein mRNAs for efficient delivery of Sec-tRNASec to the ribosome during decoding of UGA codons. However, more recent studies of SECISBP2 knockouts in mice [35] and cultured mammalian cells [36] have shown that SECISBP2 is not absolutely required for selenoprotein synthesis. Possible explanations for this observation include that a paralogue of SECISBP2 (SECISBP2L) [4] may be competent for SecRNP assembly under certain conditions or that a partially functional SecRNP may be assembled in the absence of SECISBP2. The latter observation is consistent with the observation that at least one fungal organism, G. prolifera, appears to have lost SECISBP2 while maintaining other proteins known to be required for eukaryotic selenocysteine incorporation [37]. Defining the mechanism underlying the “rescue” of SECISBP2 function may provide important clues to the molecular mechanism of SECISBP2 action.

While SECISBP2 is clearly an important protein regulating selenoprotein synthesis, many questions remain including; 1) at what stage in the SecRNP biogenesis does it interact with the SECIS element? 2) What are the molecular functions of its interaction with EEFSEC, the ribosome, and the SMN complex (discussed below)? And finally, how is occupancy of the SECIS element by several RBPs with overlapping binding sites coordinated and regulated?

What is the role of the accessory SECIS-binding proteins?

Like SECISBP2 and EEFSEC, the eukaryotic initiation factor 4a3 (EIF4A3), a member of the DEAD-box family of RNA-dependent ATPases, shuttles between the nucleus and cytoplasm. One well characterized role for EIF4A3 is to bind upstream of exon-exon junctions as an essential protein for formation of the exon junction complex (EJC) [38–41]. In this capacity, EIF4A3 serves to provide a link between pre-mRNA splicing and post-transcriptional events that include mRNA degradation by the nonsense mediated decay pathway (NMD).

In addition to its recognized role in formation of the EJC, EIF4A3 also acts as a transcript-specific repressor of selenoprotein mRNA translation [24]. During selenium deficiency, there is an increase in EIF4A3 protein, which binds selectively to SECIS elements from several selenoprotein mRNAs (e.g., GPX1 and MSRB1) that are known to be sensitive to NMD and have reduced Sec-insertion efficiencies when selenium is limiting. Mapping of the SECIS binding site of EIF4A3 indicates that it overlaps with the binding site for SECISBP2. Based on this observation, it has been proposed that EIF4A3 acts through steric hinderance to interfere with SECISBP2 binding and assembly of a functional SecRNP that can recruit EEFSEC during UGA decoding. Whether this interaction occurs in the nucleus, and possibly links SecRNP formation directly to the process of pre-mRNA splicing, or if it occurs following transport of the mRNA to the cytoplasm is not known. It is tempting to speculate that EIF4A3 interaction with the SECIS may also provide a mechanism to regulate activity of the NMD pathway for select selenoprotein mRNAs, but direct evidence for this also awaits further experimental evidence.

Another protein with SECIS binding activity is the eukaryotic ribosomal protein L30. The canonical roles of L30 include interaction with the 60S ribosomal subunit [42,43] mediated through binding to a kink-turn motif in helix 58 of the 28S rRNA, as well as binding to similar motifs in the 5’ UTR of its mRNA to auto-regulate expression [44,45]. As with other SECIS binding proteins, both ribosome bound and free L30 are found in the nucleus and cytoplasm.

As with EIF4A3, the L30 binding site has been shown to overlap the region recognized by SECISBP2 [46]. Analysis of the effects of L30 on UGA decoding indicates that increasing levels of L30 in cultured cells enhances the translational efficiency of UGA recoding [25], while the addition of free L30 to in vitro translation reactions can impede Sec insertion presumably through competition with SECISBP2 binding [46]. One possible explanation for this apparent discrepancy comes from the observation that the binding of either L30 or SECISBP2 to the SECIS element is dependent on the conformation of the essential kink-turn tandem G-A RNA motif, and further, ribosome associated L30 has a higher affinity for the SECIS element than the free protein. One model proposes that within the context of the ribosome, transient L30:SECIS interactions displace SECISBP2 and stabilizes a SECIS structural conformation that is conducive to delivery of the EEFSEC ternary complex to the ribosome for UGA decoding. Although appealing, structural evidence is lacking to support the sequence of molecular events and conformational changes that occur during the act of UGA decoding.

Finally, the SECIS binding protein, Nucleolin, is an abundant protein implicated in many cellular functions and found in multiple subcellular compartments [Reviewed in [47]]. It is the major protein in the nucleolus and, through its interactions with pre-ribosomal RNA and ribosomal proteins, plays a key role in ribosome biogenesis. While nucleolin does shuttle between the nucleus and cytoplasm, it does not interact with the mature ribosome. Cytoplasmic nucleolin has been shown in several studies to regulate mRNA stability or translation of cellular mRNAs, including the selenoproteins [27]. It has been shown that nucleolin binds to the upper basal stem of a subset of selenoprotein mRNAs that are known to be essential selenoproteins, and that knockdowns of nucleolin cause a decrease in the levels of the encoded proteins without changing mRNA levels or localization. Based on these findings, it appears that nucleolin is a positive regulator of selenoprotein translation that acts by selective binding of SECIS elements in a manner that does not compete with the other SECIS binding proteins discussed above.

What is the role of cap-hypermethylation and the SMN complex in SecRNP formation?

Eukaryotic mRNAs are characteristically modified at the 5’ end with an m7G cap that serves an important role in RNA processing, stability, and initiation of translation. For most mRNAs, the 5’ cap is recognized by the initiation factor EIF4E in the cytoplasm as a key step in translation initiation [48]. Several selenoprotein mRNAs are inefficiently recognized by EIF4E due to the fact that they are substrates of the trimethyl-guanosine synthase (TGS1) and are modified with a tri-methyl guanosine (TMG) cap similar to that acquired by small nuclear RNAs [49]. This study demonstrated that the selenoprotein mRNAs containing the TMG cap localize to the cytoplasm and are actively translated. At least one selenoprotein mRNA, Gpx1, requires TMG to support efficient protein synthesis.

TSG1 is recruited to selenoprotein mRNAs by protein:protein interactions between SECISBP2 and the ubiquitously expressed survival of motor neuron protein (SMN) complex [50]. The SMN protein, best known as the protein involved in spinal muscular atrophy, is part of an RNP assembly chaperone complex first described as being essential for assembly of small nuclear RNPs involved in splicing [51,52]. Recent findings have shown that defects in the SMN complex reduce RNP binding to messenger RNAs, affects mRNP granule formation as well as cellular mRNA localization [53]. Although no data currently supports this presumption, it is tempting to speculate that, in addition to facilitating TMG modification of several selenoprotein mRNAs, that the SMN complex may also chaperone the formation of SecRNPs involved in selenoprotein synthesis.

How does the SecRNP lead to codon specific decoding?

As a general rule, Sec-encoding UGA codons may occur in different locations in selenoprotein mRNAs and the SECIS elements may be located at any distance from the Sec codon, termination codon, or poly(A) tail. Differences in Sec insertion efficiencies when the location of a UGA codon is altered within an mRNA have generally been ascribed to local sequence contexts effects that alter termination efficiency, such as the nucleotides immediately following the UGA codon or the presence of Sec Recoding Elements (secondary structures called SREs) that reside just downstream of a subset of UGA-Sec codons [54,11,12]. Nevertheless, several selenoprotein mRNAs have been identified where the location of the UGA codon within the coding sequence is important for SECIS delivery of Sec-tRNASec. In Euplotes crassus, where UGA encodes for both Cys and Sec, several of the selenoproteins contain multiple UGA codons that are decoded as Cys or Sec in a manner that is dependent on the location of the UGA codon within the mRNA [55,56] (i.e., Sec insertion is limited to UGA codons that reside near the 3’ end of the coding sequence). In this case, the feature determining position-specific Sec insertion appears to be the 3’ UTR and is selenoprotein-specific as replacement of the 3’ UTR from one mRNA, in which Sec-insertion can normally insert Sec at any position, was sufficient to restrict Sec insertion to decoding of UGA codons near the 3’ end of the coding sequence. A deeper understanding of the overall selenoprotein mRNP and secondary structure is needed to determine how SECIS action with the ribosome is restricted spatially.

Conclusion

Ultimately, proper expression of selenoproteins is determined by RNA levels, the number of ribosomes translating each mRNA, and how efficiently the Sec-tRNASec decodes the UGA codon. The data to date suggests that these factors are inter-related and determined by the unique SecRNP that forms on each mRNA. Over the past ~30 years, the near-complete, if not complete, selenoproteome has been described, key SECIS RNA structural elements defined, and a number of RNA-binding proteins identified that bind to the SECIS elements with varying affinities. Many models have been proposed to explain general aspects of selenoprotein regulation, such as the propensity of some selenoproteins to have reduced expression under conditions of selenium deficiency, whereas the expression of other selenoproteins is well preserved [57]. Given the wide-array of selenoprotein functions it is no surprise that every selenoprotein will have evolved unique mRNA features that fine-tune expression to different biological conditions. Thus, we expect there to be features in common that are needed to support the selenoprotein synthesis machinery and variations that are required to achieve gene-specific expression levels.

We currently have only snapshots of individual players that are involved in determining the mRNA stability and translational efficiency of each selenoprotein mRNA and vague notions about how they assemble on each mRNA inside the cell to allow regulated selenoprotein mRNA maturation and selenoprotein synthesis. A common feature of the SECIS binding proteins and Sec incorporation machinery is their localization to both the nucleus and cytoplasm. These observations raise a central question: Where and when in the cell do these proteins interact with their targets? It is clear that the SecRNP must be dynamic and that RBPs play distinct roles during selenoprotein mRNA processing, maturation, translation, and ultimately, mRNA turnover. And further, that these interactions are gene-specific and regulated in response to changing cellular conditions. Future experiments to understand the mechanisms at work will require higher resolution structural studies of RNA and the SecRNP as well as in vivo temporal and spatial analysis of SecRNP formation during the different stages of the selenoprotein mRNA life cycle.

Acknowledgements

This work was supported by National Institutes of Health grants GM077073 (PRC) and GM114291 (MTH)

Footnotes

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