A plant virus replication system to assay the formation of RNA pseudotriloop motifs in RNA–protein interactions

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Abstract
A pseudotriloop is formed by transloop base pairing between the first (5′) and the fifth nucleotide in a hexanucleotide RNA loop (“hexaloop”) to subtend a triloop of nucleotides 2–4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus ε, potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo . Our data suggest that the pseudotriloop may represent a common motif in RNA–protein recognition.