Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.
We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.
Weichenrieder
O
O
European Molecular Biology Laboratory, Grenoble Outstation, France.
Kapp
U
U
Cusack
S
S
Strub
K
K
D000818:Animals; D004285:Dogs; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D018271:Signal Recognition Particle / Q000378:metabolism*
D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins; D018271:Signal Recognition Particle; C490325:Srp14 protein, mouse; C503637:Srp72 protein, mouse; D012313:RNA