Nucleic Acids Research Advance Access published online on October 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp872
Methods Online |
‘RNA walk’ a novel approach to study RNA–RNA interactions between a small RNA and its target
1The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan 52900 and 2Department of Structural Biology, Faculty of Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
*To whom correspondence should be addressed. Tel: +972 3 5318068; Fax: +972 3 7384058; Email: michaes{at}mail.biu.ac.il
Received May 20, 2009. Revised September 29, 2009. Accepted September 30, 2009.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.