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

Nucleic Acids Res. 2010 Jan;38(1):e5. doi: 10.1093/nar/gkp872. Epub 2009 Oct 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Cross-Linking Reagents
  • RNA, Protozoan
  • RNA, Ribosomal
  • RNA, Untranslated
  • Signal Recognition Particle
  • RNA, Transfer
  • aminomethyltrioxsalen
  • Ribonuclease H
  • Trioxsalen