Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein.

TLDR

EMCV mRNA is translated via internal ribosomal entry into its 5′‑nontranslated region, bypassing scanning. The study proposes that ribosomes bind directly to the initiating AUG without scanning. The IRES was mapped by in vitro translation of serially deleted EMCV 5′‑NTR RNAs generated from cDNAs. Efficient translation requires the 403–811 region, a 400‑nt upstream stem‑loop bound by a 57‑kD cellular protein, and a pyrimidine‑rich stretch near the initiation codon.

Abstract

Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5'-nontranslated region (5' NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5' NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5' or 3' end of the EMCV 5' NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A A pyrimidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.