The pRNA-assisted RNA imaging scheme (pARIS): a generalizable scaffold-based approach to facilitate cryo-EM structure determination of small RNAs

Abstract

RNA performs diverse biological functions that are often encoded in its three-dimensional structure. However, RNA-only structures represent a small fraction (~1%) of entries in the Protein Data Bank (wwPDB), limiting our knowledge of RNA structure–function relationships. The ribose-phosphate backbone of RNA is relatively flexible compared to proteins and typically forms weaker long-range tertiary interactions, resulting in structures that are less amenable to X-ray crystallography than folded proteins. Further, many functional RNAs range from 50 to 200 nt in size and are often too small for structure determination by cryo electron microscopy (cryo-EM). To facilitate RNA structure determination by cryo-EM, we present the procapsid RNA (pRNA)-assisted RNA imaging scheme (pARIS). In this approach, the RNA of interest is fused to the pRNA of bacteriophage Φ29 to enhance stability and reduce sample heterogeneity. The pRNA-linked RNA is then assembled onto the Φ29 procapsid to form a stable pentameric complex. This approach increases effective molecular mass, improves signal-to-noise ratio, and facilitates particle picking and alignments during cryo-EM imaging processing. Using pARIS, we determined a 4.8 Å structure of a 70 nt (~23 kDa) tRNA as proof of principle and a 6.7 Å structure of the liver-specific host microRNA-122 bound to its target site in the hepatitis C virus genome, demonstrating applicability to biologically relevant RNA targets.

Keywords

cryogenic electron microscopy / cryo-EM / RNA structure / RNA scaffold / bacteriophage Φ29 / pRNA / pRNA scaffold / tRNA / HCV 5’ UTR / miR-122

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