'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

• 影响指数: 19.16
• 发表时间: Jan 2010
• 来源期刊: Nucleic Acids Res
• DOI: 10.1093/nar/gkp872
• PMID: 19854950
• 文章类型: Journal Article
• 中科院分区: Q1
• 方向: 生物
• 作者列表: Lustig Y,Wachtel C,Safro M,Liu L,Michaeli S

Mesh: Base Sequence; Binding Sites; Cross-Linking Reagents / chemistry; Molecular Mimicry; Molecular Sequence Data; RNA, Protozoan / chemistry metabolism; RNA, Ribosomal / chemistry metabolism; RNA, Transfer / chemistry; RNA, Untranslated / chemistry metabolism; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease H; Signal Recognition Particle / chemistry; Trioxsalen / analogs & derivatives chemistry; Trypanosomatina / genetics

Abstract

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.