Visualizing bending of RNA structures by a bound protein using Small-Angle X-ray Scattering and Fluorescence Resonance Energy Transfer

Abstract

RNA molecules play vital roles in cellular functions, not only as facilitators of the transcription of the genetic code carried by DNA but also as non-coding molecules that promote post-transcriptional regulation of gene expression, among other functions. It is necessary to develop biophysical methods that allow visualization of the structural dynamics of non-coding RNA molecules as part of large processing complexes involving protein partners. Here, we describe how to utilize small angle x-ray scattering (SAXS) with absolute calibration and contrast variation (CV) to detect conformational changes of a primary microRNA upon binding with a part of the microprocessor complex. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA. Supporting work using single molecule Fluorescence Energy Resonance Transfer (FRET) to study the conformation of RNA duplexes bound to the protein also shows bending. Together, these studies elucidate the role of the protein DGCR8 in interacting with RNA during the early stages of microRNA processing.