In Vivo Analysis of Ribonucleoprotein Complexes Using Nucleotide Analog Interference Mapping

Abstract

Multicomponent RNA-protein complexes are essential for eukaryotic gene expression. Some, like the spliceosome, have been studied successfully in vitro using biochemical and structural approaches, but many have not been reconstituted in cell-free systems. Nucleotide analog interference mapping (NAIM) can report detailed atomic information about requirements for ribonucleoprotein particle assembly and function in living cells, providing a method to study complexes in a cellular context at a level of detail comparable to many biochemical assays. The method relies on incorporation of phosphorothioate-tagged nucleotide analogs during in vitro transcription, followed by a selection for the active population of molecules and analysis of the selected RNA sequence composition. Xenopus oocytes provide a cellular environment for selecting active molecules based on particle assembly or function. Functional group analysis of complexes assembled in vivo provides predictive models for further investigation either in vivo or in vitro as well as benchmarks for evaluating and refining biochemical and structural models.

RNA folding in living cells

“…Recently, the chemogenetic approach NAIM has been used for the first time to investigate RNA-protein interactions at atomic level in Xenopus laevis oocytes. 140,141 Random modifications of base or backbone moieties are incorporated into the transcript as nucleotide analog phosphorothioates and then microinjected in oocytes followed by identifying functional groups essential for RNP assembly in vivo. Both UV-crosslinking and NAIM could be modified to study intra-or intermolecular RNA interactions.…”