Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

Abby L Bifano,Donna M Driscoll,Tracey Ferrara,Tarik Atassi

doi:10.1186/1471-2199-14-12

Abstract

BackgroundRibosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3’ UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS.ResultsHere we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5’ side of the internal loop, the 5’ side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3’ side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets.ConclusionsThis study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs.

Highlights

Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets
We previously showed that L30 binds to Sec Insertion Sequence (SECIS) elements from multiple selenoprotein mRNAs [9], but the actual L30 binding site has not been defined
Defining the L30 binding site by RNA footprinting Eukaryotic SECIS elements are stem-loop structures containing two highly conserved motifs that are essential for Sec incorporation, namely the SECIS core and the AAR motif in an apical bulge or loop

Summary

Introduction

Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3’ UTR of mammalian selenoprotein mRNAs. The SECIS interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Based on structure probing and computer modeling, Walczak et al proposed a three-dimensional structure of the SECIS in which the RNA is kinked at the internal loop, exposing the sheared GA tandem base pairs in the SECIS core to the solvent [12]. The exact functions of SBP2 and L30 in UGA recoding have not been fully defined, and alternative models have been proposed [9,17,18]

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