Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements

Abstract

Dynamic redefinition of the 10 UGAs in human and mouse selenoprotein P (Sepp1) mRNAs to specify selenocysteine instead of termination involves two 3′ UTR structural elements (SECIS) and is regulated by selenium availability. In addition to the previously known human Sepp1 mRNA poly(A) addition site just 3′ of SECIS 2, two further sites were identified with one resulting in 10–25% of the mRNA lacking SECIS 2. To address function, mutant mice were generated with either SECIS 1 or SECIS 2 deleted or with the first UGA substituted with a serine codon. They were fed on either high or selenium-deficient diets. The mutants had very different effects on the proportions of shorter and longer product Sepp1 protein isoforms isolated from plasma, and on viability. Spatially and functionally distinctive effects of the two SECIS elements on UGA decoding were inferred. We also bioinformatically identify two selenoprotein S mRNAs with different 5′ sequences predicted to yield products with different N-termini. These results provide insights into SECIS function and mRNA processing in selenoprotein isoform diversity.

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

“…To test the function of SECIS1 and SECIS2 in vivo , we took advantage of two knockout mouse models, which are deleted for SECIS1 and SECIS2, respectively ( 32 ). As SELENOP is produced at high levels in hepatocytes for secretion into plasma, livers from wild type and SECIS deleted mice were excised and analysed by ribosome profiling and RNA-Seq.…”

“…Mice used in this study consisted of C57BL/6 (wild type) and two SECIS deletions strains, Sepp1 ΔSECIS1 and Sepp1 ΔSECIS2 , previously described ( 32 ). Whole livers were excised from male mice ( n = 3) and rapidly frozen in liquid nitrogen.…”

“…Our understanding of the mechanism by which the ribosome is reprogrammed at multiple sites in the same RNA during translation to incorporate Sec has been limited by the inability to quantify the formation of unstable termination products ( 31 ) relative to the various full-length and near full-length SELENOP proteins in vivo . To begin to address this problem, we recently described SELENOP isoforms present in serum from mice carrying genomic deletions of either Selenop SECIS1 or SECIS2 ( 32 ). Of direct biological relevance to this study is the demonstration that in primates 10–25% of Selenop mRNA naturally lacks SECIS 2 due to the utilization of a cryptic poly-(A) site located between SECIS1 and SECIS2 ( 32 ).…”

“…To begin to address this problem, we recently described SELENOP isoforms present in serum from mice carrying genomic deletions of either Selenop SECIS1 or SECIS2 ( 32 ). Of direct biological relevance to this study is the demonstration that in primates 10–25% of Selenop mRNA naturally lacks SECIS 2 due to the utilization of a cryptic poly-(A) site located between SECIS1 and SECIS2 ( 32 ). The analysis of protein products produced from the SECIS deleted mice support that the deletion of SECIS1 affects the levels of the long SELENOP isoform and deletion of SECIS2 reduces overall SELENOP abundance ( 32 ), however, the presence of multiple isoforms, uncertainties about the effects of protein processing and glycosylation on migration patterns, and the inability to identify truncated products at the first UGA limited mechanistic interpretations of the data.…”

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

“…However, further analysis of the SELENOP 3Ј UTR showed that deletion of SECIS-1 resulted in complete loss of processive Sec incorporation in favor of early termination at the second UGA, whereas deletion of SECIS-2 had little or no impact on SELENOP production (18,19). A similar study in mice showed the same result in terms of processivity, but overall production of SELENOP was reduced for the SECIS-2 deletion (20). Given that both SELENOP SECIS elements contain the conserved core motifs and are capable of Sec incorporation, we sought to determine which features within a SECIS element are required for processive Sec incorporation.…”