A novel insight into the mechanism of mammalian selenoprotein synthesis

Abstract

The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNA Sec , a 3 ′ -UTR mRNA stem-loop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNA Sec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals.

Regulation of Selenium Metabolism and Transport

“…This event prevents termination of translation and delivers selenocysteyltRNA [ser]sec to the translation machinery, allowing its selenocysteine to be inserted into the nascent protein as directed by the UGA. Additional details of this complex process and its regulation have been recognized in cultured cell and in vitro studies (53,60,64).…”

Update on Selenoprotein Biosynthesis

“…The structure from Tetrahymena thermophila (70) and Saccharomyces cerevisiae (153) are now available with sufficient resolution to unambiguous determine the 80 proteins and 5,500 RNA bases that constitute the 80S ribosome. Additionally to SBP2-ribosome interaction, evidences of a transient contact between the SECIS and at least four ribosomal proteins from the large subunits are provided by ribosomes treated with anisomycin which blocks transpeptidation (71).…”

“…In this model, it seems easier to conceive how the mRNAs are translated by several ribosomes since the SECIS-SBP2 complex is a platform to recruit and deliver the tRNA complex to the ribosome if and only if the A site is occupied by UGA codon. shown, using minimal mRNAs with "zero-length" probes in frozen translation machinery in various steps, that SECIS-SBP2, SECIS-ribosome and SBP2-ribosome interactions are highly dynamic during the different elongation phases of selenoprotein mRNA translation (71).…”

Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation

“…Post-labeling of RNAs isolated from the purified complexes was performed in general according to [34]. Briefly, 48S and 80S ribosomal complexes (obtained from 100 l of RRL each) were purified as described above by centrifugation in sucrose gradient with subsequent ethanol precipitation of ribosomal fractions.…”

“…Finally, 80S elongation complexes (80S EC) modeling a state immediately after the accommodation of aminoacyl-tRNA to the A site before transpeptidation were assembled in RRL with mRNA MIII and HCV IRES FL using antibiotic anisomycin that blocks peptide bond formation[35] (Figure 2). The complexes were obtained exactly according to protocols described in[34] where their nature has been examined by specialassays. Here we confirmed the composition of the model complexes, analyzing the products of post-labeling of RNAs isolated from these complexes by denaturing PAGE.…”