The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA

Abstract

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.

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

“…After chemical probing or cross-linking procedures, 48S and/or 80S complexes were purified by centrifugation in 15-30 % sucrose gradient in buffer B (20 mM HEPES-KOH, pH 7.8, 100 mM KOAc, 10 mM 2-mercaptoethanol and 3 mM Mg(OAc) 2 ) as described before [31,32]. The mixtures layered onto the gradient were centrifuged (65000g, 17.5 h, rotor SW40) in an…”

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

“…Notably, the apical loop of H89 undergoes conformational rearrangements as well when the ribosome binds the SelenoCysteine Insertion Sequence (SECIS) binding protein 2 (SBP2) (Kossinova et al 2014), a key player in selenoprotein synthesis utilizing recoding of the UGA stop codon as a selenocysteine (Sec) codon (e.g., see Bulteau and Chavatte 2015). The functions of the protein are based on its ability to interact with SECIS present in the 3 ′ UTR of all eukaryotic selenoprotein mRNAs and with the ribosome-in particular, with helix ES7L-E of the 28S rRNA (Kossinova et al 2014 and references therein).…”

“…SBP2 bound to SECIS of selenoprotein mRNA has been suggested to provide delivery of SectRNA Sec (within its ternary complex with GTP and a specific elongation factor eEFSec) to the ribosomal A site programmed with UGA codon and its subsequent accommodation to this site ). The rearrangements caused by SBP2 binding to the ribosome involve H89 apex sites (riboses of U4419 and C4421 [Caban and Copeland 2012] and nucleotide bases of A4414 and A4422 [Kossinova et al 2014]) that are not implicated in structural changes induced by release factors binding (Table 1). One can assume that SPB2-induced rearrangements in the H89 apex prevent the accommodation corridor from opening by eRF1.…”

“…Results obtained by means of chemical footprinting were in general compatible with respective structural data resulted from cryo-EM and X-ray crystallography studies. Utilizing this strategy, the pivotal protein component of the selenoprotein synthesis machinery, the so-called SBP2, which remained unresolved using cryo-EM and X-ray crystallography analysis, was mapped on the 60S subunit structure (Kossinova et al 2014). Moreover, conformational rearrangements induced by binding of HCV IRES RNA to the 40S subunit ) and of SBP2 to the 80S or 60S ribosome (Kossinova et al 2014) have been revealed with the use of chemical footprinting.…”

Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements

“…This binding is likely weaker than SECIS 1 [84], raising the potential for a tethering role for linking the SECIS-associated complex to the vicinity of UGA 1. However, while SBP2 is really important for Sepp1 expression [29,30], it is not essential and intriguingly plays a major role in stabilizing Sepp1 mRNA [31]. Whether the intron junction is relevant to this is unknown.…”