Solution Structure Determination and Biophysical Studies of 7SK RNP and 7SL SRP RNAs

Abstract

Small noncoding RNAs perform integral roles in eukaryotic lifecycles, particularly the 7SK snRNA, which is responsible for RNA Polymerase II transcription modulation and progression when interacting with P-TEFb, and the 7SL RNA involved in Signal Recognition Particle mediation of co-translational activities of endoplasmic reticulum-bound proteins. These RNAs retain important secondary structures that interact with proteins involved in regulating transcription and translation. RNA-protein interactions involving the RNA stem-loops have been previously characterised using chemical probing techniques, Cryo-Electron Microscopy, and Nuclear Magnetic Resonance. However, complete three-dimensional structures of the full-length 7SK and 7SL have not been resolved, limiting our understanding of these RNAs’ tertiary landscapes and mechanisms. Our study bridges this gap in knowledge by using Small-Angle X-ray Scattering and coarse-grained computational modelling of previously determined secondary structures through SimRNA to produce full-sequence, three-dimensional atomistic models of both 7SK and 7SL RNAs. We employed size exclusion chromatography coupled with light scattering and Circular Dichroism Spectroscopy to verify RNA size and compare previously identified secondary structures in solution. We additionally employed all-atom, structure-based potential simulations to generate optimised models within our calculated SAXS envelopes. 7SK’s total morphology is thus presented as a highly versatile structure whose well-defined stem-loops interact with each other in three-dimensional space. 7SL RNA is presented as a tightly wound and somewhat rigid structure, with significant base-pairing features in its Alu domain, whereupon it forms likely scaffolds for signal recognition peptide formation.