'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.
In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by 'RNA walk' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that 'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.
Lustig
Yaniv
Y
The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel.
Wachtel
Chaim
C
Safro
Mark
M
Liu
Li
L
Michaeli
Shulamit
S
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D016053:RNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry; D022661:RNA, Untranslated / Q000737:chemistry* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H; D018271:Signal Recognition Particle / Q000737:chemistry*; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000737:chemistry; D014351:Trypanosomatina / Q000235:genetics
D003432:Cross-Linking Reagents; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D022661:RNA, Untranslated; D018271:Signal Recognition Particle; D012343:RNA, Transfer; C018822:aminomethyltrioxsalen; D016914:Ribonuclease H; D014307:Trioxsalen