In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp).
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.
Bortolin
Marie-Line
ML
Laboratoire de Biologie Moléculaire Eucaryote, UMR5099 du CNRS, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
Bachellerie
Jean-Pierre
JP
Clouet-d'Orval
Béatrice
B
D019843:Archaeal Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D011485:Protein Binding; D043903:Pyrococcus abyssi / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000235:genetics / Q000378:metabolism*; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*
D019843:Archaeal Proteins; D012322:RNA Precursors; D019642:RNA, Archaeal; D012364:RNA, Transfer, Trp; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins