In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNATrp

Abstract

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre‐tRNA^Trp represents an outstanding specimen able to guide in cis, instead of in trans, two 2′‐O‐methylations in the pre‐tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge–helix–bulge (BHB) splicing motif, while the RNA‐guided methylation and pre‐tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre‐tRNA and tested its site‐specific methylation activity. Recognition by L7Ae of hallmark K‐turns at the C/D and C′/D′ motifs appears as a crucial assembly step required for subsequent binding of a Nop5p–aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p–aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis‐acting system to form guide RNA duplex(es) relative to classical trans‐ acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non‐canonical stem of the K‐turn, suggest that binding of Nop5p–aFib to the pre‐tRNA–L7Ae complex might direct transition from a splicing‐competent structure to an RNA conformer displaying the guide RNA duplexes required for site‐specific methylation.