The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA.
SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem-loop in the 3' UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg, France.
Olga
O
Kossinova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Alexey
A
Malygin
Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg, France.
Alain
A
Krol
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Galina
G
Karpova
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012339:RNA, Ribosomal, 28S / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D051140:Selenoproteins / Q000096:biosynthesis*; D012689:Sequence Homology, Nucleic Acid
D012339:RNA, Ribosomal, 28S; D016601:RNA-Binding Proteins; C121530:SECISBP2 protein, human; D051140:Selenoproteins
SECIS-binding protein 2; chemical probing; cross-linking approach; mammalian ribosome; selenocysteine incorporation