Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.

Franck Martin,Lydia Prongidi-Fix,S Kundhavai Natchiar,Jean-François Ménétret,Quentin Vicens,Alexander G Myasnikov,Angelita Simonetti,Gilbert Eriani,Bruno P Klaholz

doi:10.1038/ncomms12622

Abstract

Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon.

Highlights

Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon
Mouse h4 mRNA/rabbit 80S complexes were assembled in rabbit reticulocyte lysate and stalled in the initiation state by cycloheximide and hygromycin B that prevent the elongation at translocation steps
The structure contains an initiator tRNA accommodated in the peptidyl (P) site and a ternary complex of eEF1A-tRNA localized in the factor-binding site (Fig. 1a) reminiscent of a late 80S translation initiation complex in which codon recognition has occurred

Summary

Introduction

Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. Variations of the Kozak sequence can lead to initiation at downstream AUG triplets by leaky scanning[7] Deviations from this classical model exist, for example, viral mRNAs that contain 50 untranslated region (UTR) internal ribosomal entry sites (IRES) often require only a subset of the initiation factors to hijack the ribosome, as visualized by several cryo-EM structures[8,9,10,11,12,13]. The TWJ is located 19 nucleotides downstream from the AUG codon, and is flanked by a weak Kozak sequence (with a U in position þ 4) and a double stem-loop structure called eIF4E-sensitive element (4E-SE) (Supplementary Fig. 1)[14] These specific RNA structures tether the translation machinery directly on the first AUG initiation codon of h4 mRNA, regardless of the presence of a second in-frame initiation codon. We localize the folded h4 mRNA TWJ domain on the rabbit ribosome using cryo-EM and show by toe-printing and mutational analysis that h4 mRNA exhibits shortly after the start codon a sequence complementary to the 18S rRNA sequence that helps mRNA binding and proper AUG positioning

Methods

Results

Conclusion