Regulation of selenocysteine insertion in eukaryotes: Functional parameters of the SECIS element and in vivo efficiency of selenoprotein synthesis.

摘要

Selenoproteins are proteins which contain the unusual amino acid selenocysteine, the selenium-containing analog of serine and cysteine. Selenocysteine is inserted during translation at in-frame UGA codons. In eukaryotes, a cis-acting element in the 3' untranslated region (UTR) of the selenoprotein mRNA, termed the selenocysteine insertion sequence (SECIS), is required for the recoding of UGA-termination codons to UGA-selenocysteine codons. Here, a minimal functional eukaryotic SECIS element is defined for the first time as a 42-nucleotide stem-loop structure. This structure must be placed within the 3' UTR, as, when it is at least partially translated it functions poorly, drawing an important distinction from selenoprotein synthesis in E. coli In addition, the SECIS element must lie more than 51--60 nucleotides downstream of a UGA codon in order to specify selenocysteine insertion. The SECIS element also contains a core region consisting of invariant nucleotides. Through extensive mutational analysis, it is reported here that while the nucleotides outside the core are not critical for the function of the SECIS element, their identities can influence the effect of mutations to the invariant core nucleotides. Similarly, while nucleotides below the core are not conserved, nor are they required for SECIS activity since they are beyond the minimal functional element, the open structure that they are predicted to form is crucial for the SECIS element function.; Polysome analysis allows the determination of the number of ribosomes, on average, bound to a particular mRNA in vivo. This technique was used to assess the ribosome association with endogenous selenoprotein mRNAs, as compared to their transiently transfected UGA-to-UAA termination and UGA-to-UGU cysteine mutant counterparts. The results show that selenoprotein mRNAs are not efficiently translated and that the UGA codon acts as an impediment to efficient ribosome loading and/or transit across selenoprotein mRNAs.