A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors.

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred.

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay.

Assembly of 48S translation initiation complexes from purified components with mRNAs that have some base pairing within their 5' untranslated regions.

The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly.

Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism.