Summary auto-generated
This study investigates how influenza A virus M1 protein binds to RNA. Researchers used chemical cross-linking techniques with trans-DDP and APG to identify the specific M1 peptide sequence that contacts RNA and the RNA sequences involved in binding. They found that M1 binds to RNA without sequence specificity, meaning it can bind to various RNA types indiscriminately. The actual RNA-binding domain was identified as a peptide region containing the RKLKR nuclear localization signal (NLS) at amino acid positions 101-105. Site-directed mutagenesis confirmed that mutating all basic residues in this region abolished RNA-binding activity. Interestingly, a mutant lacking RNA-binding capacity retained partial transcription-inhibition activity, suggesting these two functions are related but not identical. The zinc-binding sequence previously proposed to be involved in RNA-binding was shown to be unnecessary for this interaction. These findings demonstrate that the M1 protein's nuclear localization signal serves a dual function as an RNA-binding domain, explaining the overlap between nuclear import and RNA-binding activities observed in many proteins.
Key findings
- M1 protein binds RNA through its nuclear localization signal (RKLKR sequence at positions 101-105), not through sequence-specific recognition
- M1 binds to RNA with no sequence specificity, binding similarly to various RNA types, tRNA, and even double-stranded DNA
- The zinc-binding sequence previously implicated in RNA-binding is not essential for RNA-binding or transcription-inhibition activities
- A mutant M1 lacking RNA-binding activity retained approximately 50% of its transcription-inhibition ability, indicating these activities are distinct but related
- The arginine-rich NLS motif represents a classic RNA-binding signature common to proteins with overlapping nuclear import and RNA-binding functions
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Abstract
The RNA-binding activity of influenza A virus M1 protein was studied by cross-linking the protein to viral RNA followed by sequence analysis of the oligoribonucleotide bound to the protein as well as sequence analysis of the M1 peptide bound to the RNA. M1 was found to bind to RNA without any RNA sequence specificity, as verified in a series of filter-binding experiments using a large variety of nucleic acids including DNA. The peptide sequence that bound to the RNA was the RKLKR nuclear localization signal of M1. Site-specific mutagenesis of recombinant M1 showed that most of the basic residues in that region had to be mutated in order to inhibit RNA-binding. We also constructed an M1 mutant that no longer bound to RNA but which was still able to inhibit the in vitro transcription activity of isolated viral ribonucleoprotein, albeit to a lower extent. Mutation of the zinc- binding sequence had no effect on RNA-binding or transcription- inhibition activity.