Nucleic Acids Research, 2003, Vol. 31, No. 22 6524-6535
© 2003 Oxford University Press
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In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNATrp
Laboratoire de Biologie Moléculaire Eucaryote, UMR5099 du CNRS, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France
*To whom correspondence should be addressed. Tel: +33 5 61 33 59 07; Fax: +33 5 61 33 58 86; Email: clouet{at}ibcg.biotoul.fr
Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNATrp 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.
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