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

Abstract

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Seccontaining 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.

A Summary of New Findings on the Biological Effects of Selenium in Selected Animal Species—A Critical Review

“…The incorporation of selenocysteine into selenoproteins utilizes an unique mechanism that requires decoding of the codon UGA [ 63 ] in mRNA, normally involved in the termination of translation [ 64 , 65 , 66 ]. The UGA recoding is strictly dependent on an RNA stem-loop structure, the selenocysteine insertion sequence (SECIS), which is found in the 3′ untranslated region (3′ UTR) of eukaryotic selenoprotein messages [ 67 , 68 ].…”

Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress

“…Influence of diphthamide on the biosynthesis of selenocysteine containing proteins was deduced from observations that diphthamide deficiencies (i) reduce translation of recombinant full length selenoproteins, (ii) affect the transcription of selenoproteins, (iii) render cells hypersensitive to oxidative stress and (iv) increase tolerance towards selenite. The basic principle of selenocysteine incorporation at UGA codons accompanied by SECIS elements is conserved in all organisms [20] , [21] , [22] . Incorporation processes, however, differ between eukaryotes and archaebacteria on one side, and eubacteria on the other side.…”

“…Selenocysteine containing proteins are a small group of proteins whose translation involves a special incorporation process at the elongation-vs-termination decision point [18] , [19] , [20] , [21] , [22] , [23] . Their mRNAs harbor UGA codons which are not recognized as termination signals but instead decoded as selenocysteine, dependent on presence of 3′UTR-SECIS elements.…”

“…Their mRNAs harbor UGA codons which are not recognized as termination signals but instead decoded as selenocysteine, dependent on presence of 3′UTR-SECIS elements. Most selenoproteins are involved in redox-processes to manage or detoxify oxidative stress [21] , [22] , [23] and oxidative stress is a known trigger of NFkB activation [24] , [25] , [26] . Therefore, we have analyzed if diphthamide deficiency influences the expression of selenoproteins and if it affects (as downstream consequence) cellular sensitivity towards oxidative stress.…”

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity

“…The conserved cysteine residues are represented in boxes with red borders; they are important for the binding capacity of the SECIS region to selenoprotein mRNAs (Bubenik et al, 2015). A search of the National Center for Biotechnology Information (NCBI) conserved domains database (CDD) hit the binding domain of the ribosomal protein 7Ae/L30e/S12e/Gadd45 family and the RNA binding domain in the overlapped region shaded grey in the alignment (Fig.…”