Functional Analysis of Noncoding RNAs in Trypanosomes: RNA Walk, a Novel Approach to Study RNA–RNA Interactions Between Small RNA and Its Target
The recent discovery of thousands of small noncoding RNAs (ncRNAs), in many different organisms, has led to the need for methods to study their function. One way to help understand their function is to determine what other RNAs interact with the ncRNAs. We have developed 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. Domains carrying the cross-linked adducts are less efficiently amplified than domains that are not cross-linked. Real-time PCR is used to quantify the results. Further mapping of the interactions is performed by primer extension to determine the exact cross-linked adduct.
Key wordsRNA walk RNA–RNA interactions Cross-linking Affinity selection Trypanosomes
This research was supported by a grant from the Israel–US Binational Science Foundation (BSF), and by an International Research Scholar’s Grant from the Howard Hughes Foundation to S.M. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases.
- 1.Mattick, J. S., and Makunin, I. V. (2006) Non-coding RNA, Hum Mol Genet 15 Spec No 1, R17–R29.Google Scholar
- 10.Brun, R., and Schonenberger, M. (1979) Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication, Acta Trop 36, 289–292.Google Scholar