Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element.
SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons. In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region. Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function. These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously. We also define base pairing requirements in the stem of this element. In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function. Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.
Martin
G W
GW
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA.
Harney
J W
JW
Berry
M J
MJ
D000818:Animals; D001483:Base Sequence; D007453:Iodide Peroxidase / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D051149:Selenoprotein P; D051140:Selenoproteins; D017421:Sequence Analysis
D011506:Proteins; D012333:RNA, Messenger; D051149:Selenoprotein P; D051140:Selenoproteins; D017279:Selenocysteine; D007453:Iodide Peroxidase