A yeast RNA-hybrid system for the detection of RNA–RNA interactions in vivo

Abstract

RNA-RNA interactions play a crucial role at many different levels of the cellular metabolism such as plasmid replication control, viral encapsidation, or transcriptional and translational regulation. Therefore, methods are necessary to investigate the molecular determinants of given interactions, including their stabilities, or to screen for new interacting partners. We designed an RNAhybrid system in S. cerevisiae, based on the yeast three-hybrid system. In this setup, the activation of a reporter gene is dependent on the interaction of two RNAs. A loop-loop interaction similar to the dimerization initiation site of the HIV genome was used as a model system, demonstrating that in this novel RNA-hybrid system only cognate RNAs promote the activation of the reporter gene. Levels of reporter activation correlate well with interaction stabilities determined in vitro by UV melting analyses, suggesting that conditions used for the analysis of in vitro structural stabilities translate well into the intracellular environment. Furthermore, the system was applicable for a screen against a test library. Nine out of ten selected clones were identified as predicted interaction partners for the bait RNA. In summary, we present a yeast reporter system depending on RNA-RNA interactions, which can be used alternatively for analysis of known interactions or for screening libraries in search for new interaction partners.

Advances and challenges towards the study of RNA-RNA interactions in a transcriptome-wide scale

“…Cellular methods to investigate certain RRIs also have been designed. For instance, an RNA-hybrid system in Saccharomyces cerevisiae has been developed to detect a certain RRI by using a reporter gene whose activation depends on the interaction of two RNAs [32,33] ( Figure 1E). Another way to validate RRIs is to construct mutants that disrupt the putative base-pairing, and more importantly, rescue mutants that restore the disrupted basepairings by complementary mutations.…”

The Chemical Biology of Aptamers

“…[165,[169][170][171][172] Recently, selection approaches based on the yeast three hybrid system have been described to assess the in vivo properties of in vitro selected RNA aptamers and to improve the activities of aptamers in vivo. [173][174][175][176][177] Lipid-based transfection experiments, which are used for the transmembrane location of plasmids and siRNA molecules, can also be used to deliver aptamers into the cytoplasm ( Figure 6). [178] Other methodologies that might be useful for delivering aptamers inside cells include microinjection, receptor-mediated internalization, and the construction of transgenic animals expressing the aptamer under the control of conditional promoters, as shown in a landmark study by Lis and co-workers.…”

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

“…Because the amino acids that recognize adenine, uracil, and guanine were discovered previously, artificial PUF proteins can be created to bind specific mRNA sequences for translation control (179), a method that could become complementary to RNA interference (163). Alternative RNA three-hybrid setups have enabled the identification of trimeric protein-RNA complexes (54,411) and RNA-RNA interactions (515) and the design of transcription-activating RNA stretches (687). Finally, an ingenious bacterial onehybrid method is available for detection of heterologous RNAprotein interactions, based on lacZ expression upon relief from antitermination (704).…”