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Updated: Sep 23, 2026
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
Published on: September 30, 2016
The internal ribosome entry site (IRES) contained within the RNA-binding motif protein 3 (Rbm3) mRNA is composed of
Stephen A Chappell1, Vincent P Mauro
1Department of Neurobiology, The Scripps Research Institute, and The Skaggs Institute for Chemical Biology, La Jolla, California 92037, USA.
Abstract:
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.
Insights
Short nucleotide sequences can initiate translation, similar to viral internal ribosome entry sites (IRESes). Researchers identified a modular IRES in Rbm3 mRNA, with a 22-nucleotide module binding directly to ribosomal subunits to facilitate translation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Internal ribosome entry sites (IRESes) are crucial for cap-independent translation initiation in viral and cellular mRNAs.
- While viral IRESes are large, evidence suggests shorter sequences can also mediate internal translation initiation.
- The Rbm3 mRNA 5' leader contains a 720-nucleotide sequence with potential regulatory elements for translation.
Purpose of the Study:
- To investigate the modularity and functional cis-acting elements within the Rbm3 mRNA 5' leader IRES.
- To identify the minimal sequence required for IRES activity and its mechanism of action.
- To determine the protein and ribosomal interactions of the identified IRES elements.
Main Methods:
- Deletion and mutational analyses of the Rbm3 mRNA 5' leader sequence.
- In vitro assays to test IRES function, including assessment of transcriptional and splicing activities.
- Protein binding studies using cytoplasmic proteins and 40S ribosomal subunits.
Main Results:
- The Rbm3 mRNA IRES is modular, comprising at least 9 cis-acting sequences, including a 22-nt IRES module, a 10-nt enhancer, and two inhibitory sequences.
- The 22-nt sequence functions as an IRES independently and does not act as a promoter, enhancer, or splice site.
- Four cis-acting sequences bind specifically to distinct cytoplasmic proteins, and the 22-nt IRES module binds directly to 40S ribosomal subunits.
Conclusions:
- The Rbm3 mRNA 5' leader contains multiple cis-acting elements that mediate and modulate internal translation initiation.
- A minimal 22-nucleotide IRES module facilitates translation by directly interacting with 40S ribosomal subunits.
- These findings reveal a novel mechanism for cap-independent translation initiation mediated by short IRES elements.
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