canonical pathway for selenocysteine insertion is dispensable in Trypanosomes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 13; pp. 5088 - 5092
• Main Authors: Aeby, Eric, Palioura, Sotiria, Pusnik, Mascha, Marazzi, Janine, Lieberman, Allyson, Ullu, Elisabetta, Söll, Dieter, Schneider, André
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 31.03.2009; National Acad Sciences
• Subjects: Animals; Archaea; Biological Sciences; Cell lines; Enzymes; Eukaryotic cells; Gels; in vitro studies; Metabolic Networks and Pathways; Mice; mutants; Parasitism; phosphorylation; Phosphotransferases - metabolism; RNA; RNA interference; RNA, Transfer, Amino Acid-Specific - metabolism; RNA, Transfer, Amino Acyl - metabolism; Selenium; selenocysteine; Selenocysteine - metabolism; Selenoproteins; Selenoproteins - biosynthesis; serine; serine-tRNA ligase; Transfer RNA; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; Trypanosoma brucei brucei - metabolism; viability
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.0901575106

The micronutrient selenium is found in proteins as selenocysteine (Sec), the 21st amino acid cotranslationally inserted in response to a UGA codon. In vitro studies in archaea and mouse showed that Sec-tRNASec formation is a 3-step process starting with serylation of tRNASec by seryl-tRNA synthetase (SerRS), phosphorylation of serine to form phosphoserine (Sep)-tRNASec by phosphoseryl-tRNASec kinase (PSTK), and conversion to Sec-tRNASec by Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic selenoprotein synthesis has been lacking. Here, we present an analysis of Sec-tRNASec formation in the parasitic protozoon Trypanosoma brucei in vivo. Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, demonstrating the essentiality of both enzymes for Sec-tRNASec formation. Growth of the 2 knockout strains was not impaired; thus, unlike mammals, trypanosomes do not require selenoproteins for viability. Analysis of conditional RNAi strains showed that SerRS, selenophosphate synthase, and the Sec-specific elongation factor, EFSec, are also essential for selenoprotein synthesis. These results with T. brucei imply that eukaryotes have a single pathway of Sec-tRNASec synthesis that requires Sep-tRNASec as an intermediate.