Multiple RNA structures affect translation initiation and UGA redefinition efficiency during synthesis of selenoprotein P

Abstract

Gene-specific expansion of the genetic code allows for UGA codons to specify the amino acid selenocysteine (Sec). A striking example of UGA redefinition occurs during translation of the mRNA coding for the selenium transport protein, selenoprotein P (SELENOP), which in vertebrates may contain up to 22 in-frame UGA codons. Sec incorporation at the first and downstream UGA codons occurs with variable efficiencies to control synthesis of full-length and truncated SELENOP isoforms. To address how the Selenop mRNA can direct dynamic codon redefinition in different regions of the same mRNA, we undertook a comprehensive search for phylogenetically conserved RNA structures and examined the function of these structures using cell-based assays, in vitro translation systems, and in vivo ribosome profiling of liver tissue from mice carrying genomic deletions of 3′ UTR selenocysteine-insertion-sequences (SECIS1 and SECIS2). The data support a novel RNA structure near the start codon that impacts translation initiation, structures located adjacent to UGA codons, additional coding sequence regions necessary for efficient production of full-length SELENOP, and distinct roles for SECIS1 and SECIS2 at UGA codons. Our results uncover a remarkable diversity of RNA elements conducting multiple occurrences of UGA redefinition to control the synthesis of full-length and truncated SELENOP isoforms.

Processive incorporation of multiple selenocysteine residues is driven by a novel feature of the selenocysteine insertion sequence

“…The detection of early termination products by SECIS-1 alone supports earlier studies indicating that although SECIS-2 is not essential for Sec incorporation, it primarily affects decoding efficiency at the first UGA codon (19,39). The first UGA has been shown to be the rate-limiting step in selenoprotein translation, and recent ribosome profiling of SELENOP mRNA clearly identified pausing at the first UGA (39). Thus, slow decoding at the first UGA by SECIS-2 may be essential to ensure full-length protein synthesis.…”

“…On the other hand, the SECIS-1 alone mutant produced a full-length protein similar in efficiency to the WT 3Ј UTR but also produced some intermediate products that correspond to termination between the second UGA and the last (17th) UGA. The detection of early termination products by SECIS-1 alone supports earlier studies indicating that although SECIS-2 is not essential for Sec incorporation, it primarily affects decoding efficiency at the first UGA codon (19,39). The first UGA has been shown to be the rate-limiting step in selenoprotein translation, and recent ribosome profiling of SELENOP mRNA clearly identified pausing at the first UGA (39).…”

The algal selenoproteomes

“…SELENOP is a selenoprotein with multiple Sec residues. Its multiple Sec structure helps it to serve as storage and transport equipment in different cells or tissues in the multicellular animals [59]. Interestingly a multi-Sec selenoprotein family was also The composition of selenoproteomes in species with closely evolutionary relationships is similar.…”

The Selenium Transport Protein, Selenoprotein P, Requires Coding Sequence Determinants to Promote Efficient Selenocysteine Incorporation

“…This raises a fundamental mechanistic question about processive Sec incorporation: What differentiates Sec incorporation at the first UGA versus downstream UGA codons? Recently a direct comparison of in vitro synthesis of wild type human SELENOP versus an artificial luciferase construct with 10 UGA codons positioned exactly as found in human SELENOP but placed in a non native context was performed (35). In this case, the artificial selenoprotein resulted in dramatically lower efficiency and processivity, thus strongly implicating a role for coding region sequence.…”

“…Also, bioinformatic analysis identified four structural elements in the coding region termed as initiation stem loop (ISL) and Sec Redefinition Elements -SRE1, SRE2 and SRE3. In a reporter construct, the ISL and SRE1 influenced translation initiation and Sec incorporation at the first UGA, respectively while the role of SRE2 and 3 are yet to be determined (35). And finally, since all artificial constructs with multiple Sec codons have been analyzed using SELENOP SECIS elements, it seems most likely that although SECIS elements are sufficient for Sec incorporation, the coding region sequence is critical to modulate efficiency and processivity.…”