The Internal Ribosome Entry Site (IRES) Contained within the RNA-binding Motif Protein 3 (Rbm3) mRNA Is Composed of Functionally Distinct Elements

• Published in: The Journal of biological chemistry 2003-09, Vol.278 (36), p.33793-33800
• Main Authors: Chappell, Stephen A., Mauro, Vincent P.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Motifs; Animals; Base Sequence; Binding Sites; Cell Line; Cell-Free System; Cytoplasm - metabolism; DNA - metabolism; DNA Mutational Analysis; Gene Deletion; Humans; Luciferases - metabolism; Mice; Molecular Sequence Data; Mutation; Open Reading Frames; Protein Binding; Protein Biosynthesis; Rats; Ribosomes - chemistry; Ribosomes - metabolism; RNA, Messenger - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - physiology; Transfection; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M303495200

Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, we performed deletion and mutational analyses of an IRES contained within the 720-nucleotide (nt) 5′ leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. These cis-acting sequences include a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. The 22-nt sequence was shown to function as an IRES when tested in isolation, and we demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site. The activities of all 4 cis-acting sequences were further confirmed by their mutation in the context of the full IRES. Interestingly, one of the inhibitory cis-acting sequences is contained within an upstream open reading frame (uORF), and its activity seems to be masked by translation of this uORF. Binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins. In addition, the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. The results demonstrate that different types of cis-acting sequences mediate or modulate translation of the Rbm3 mRNA and suggest that one of the IRES modules contained within the 5′ leader facilitates translation initiation by binding directly to 40 S ribosomal subunits.