Analysis of Protein–RNA Complexes Involving a RNA Recognition Motif Engineered To Bind Hairpins with Seven- and Eight-Nucleotide Loops
2013
Blakeley, Brett D. | Shattuck, Jenifer | Coates, Melissa B. | Tran, Evelyn | Laird-Offringa, Ite A. | McNaughton, Brian R.
U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.
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