Biology subjects

Feyrer, H.

Publications and source records attributed to Feyrer, H..

2 recordsLinked to original sources

bioRxiv2022

Mass spectrometry of RNA-binding proteins during liquid-liquid phase separation reveals distinct assembly mechanisms and droplet architectures

Phase separation of heterogeneous ribonucleoproteins (hRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility-mass spectrometry, and molecular dynamics simulations. We used a phase separation-compatible spider silk domain and pH changes to control the self-assembly of the hRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with phase separation. We find that NT*-FUS monomers undergo an unfolded-to-globular transition, whereas NT*-TDP-43 oligomerizes into partially disordered dimers and trimers. NT*-hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over phase separation. The divergent assembly mechanisms result in structurally distinct complexes, indicating differences in RNA processing and translation depending on biological context.

biophysics↗

bioRxiv2022

Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods pose as an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5 position, which allows 5-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

biochemistry↗