title: 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.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Kossinova
authors.forename: Alexey
authors.initials: A
authors.lastname: Malygin
authors.forename: Alain
authors.initials: A
authors.lastname: Krol
authors.forename: Galina
authors.initials: G
authors.lastname: Karpova
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D051140:Selenoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D012333:RNA, Messenger; D012345:RNA, Transfer, Amino Acid-Specific; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C121530:SECISBP2 protein, human; D051140:Selenoproteins; C036928:tRNA, selenocysteine-
keywords: SECIS-binding protein 2; cross-linking approach; mammalian ribosome; selenocysteine incorporation; selenocysteine insertion sequence