Fusion RNAs in crystallographic studies of double-stranded RNA from trypanosome RNA editing.

Abstract

Head-to-head fusions of two identical double-stranded fragments of RNA can be designed to self-assemble from a single RNA species and form a double-stranded helix with a twofold rotation axis relating the two strands. These symmetrical RNA molecules are more likely to crystallize without end-on-end statistical packing disorder because the two halves of the molecule are identical. This approach can be used to study many fragments of double-stranded RNA or many isolated helical domains from large single-stranded RNAs that may not yet be amenable to high-resolution studies by crystallography or NMR. We used fusion RNAs to study one (the U-helix) of three functional domains formed when guide RNA binds to its cognate pre-edited mRNA from the trypanosome RNA editing system. The U-helix forms when the 3' oligo(U) tail of the guide RNA (gRNA) binds to the purine-rich, pre-edited mRNA upstream from the current RNA editing site. Fusion RNAs 16-and 32-base pairs in length formed crystals that gave diffraction to 1.37 and 1.05 Å respectively. We provide the composition of a fusion RNA crystallization screen and describe the X-ray data collection, structure determination, and refinement of the crystal structures of fusion RNAs.