RNA systems for NMR studies in vitro and in vivo

NMR spectroscopy is an excellent tool to study the structure-function relationship of RNA. Such measurements are usually performed in vitro, which requires large amounts of isotope- labeled sample in high purity and can give access to individual atoms, structure of the molecule and conformational dynamics. This is contrasted by measurements in living cells, where researchers struggle with low signal intensity, line-broadening and rapid sample degradation. In this work, we developed sample preparation methods for NMR studies to expand the range of RNA constructs that are accessible for NMR studies in vitro and in cells. Firstly, we improved yield and purity of in vitro transcription of short RNA constructs by transcribing several repeating target sequences from a tandem template, and cleaving them to the target length with RNase H. This abolishes issues with suboptimal initiation sequences and creates higher purity due to the high sequence-specificity of RNase H guided by a chimeric oligo. We demonstrated the high yield and purity of several such RNA molecules and incorporated the protocol into a workflow for studies of conformational dynamics with relaxation dispersion NMR. Secondly, we demonstrated the site-specific incorporation of a 13 C/15 N-labeled adenosine into a 46 nt RNA molecule with the use of purely enzymatic methods. Such site-specific labeling is an effective approach to overcome resonance overlap in larger RNAs, which can preclude further structural and dynamics studies. We showed the facile production of such a sample and reported on a second conformation which would in a uniformly labeled sample be hidden by overlapping resonances. Lastly, we furthered method development for in-cell NMR methods by exploring transfection strategies, cell culture methods and RNA systems. We adapted a protocol for the production of circular RNA at high concentration in HEK293T cells to generate the first in-cell NMR spectra of intracellular expressed RNAs. Furthermore, we produced the same circular RNAs by in vitro transcription and ligation to assess their improved stability against cellular exonucleases. As circular RNA model systems, we used the fluorescent aptamer Broccoli and a small hairpin RNA, called GUG, which proved useful for relaxation dispersion NMR measurement previously. The expression of both circular constructs at was possible at micromolar concentration in HEK283T cells and both constructs could be transcribed and circularized in vitro. In-cell NMR of the expressed circular RNA did however not yield detectable signals, indicating that either the intracellular concentration is too low, or the location of the expressed RNA precludes free tumbling

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures

Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) 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 an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs 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 liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA

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 represent 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

Toward Precise Interpretation of DEER-Based Distance Distributions: Insights from Structural Characterization of V1 Spin-Labeled Side Chains

Pulsed electron paramagnetic resonance experiments can measure individual distances between two spin-labeled side chains in proteins in the range of ∼1.5–8 nm. However, the flexibility of traditional spin-labeled side chains leads to diffuse spin density loci and thus distance distributions with relatively broad peaks, thereby complicating the interpretation of protein conformational states. Here we analyzed the spin-labeled V1 side chain, which is internally anchored and hence less flexible. Crystal structures of V1-labeled T4 lysozyme constructs carrying the V1 side chain on α-helical segments suggest that V1 side chains adopt only a few discrete rotamers. In most cases, only one rotamer is observed at a given site, explaining the frequently observed narrow distance distribution for doubly V1-labeled proteins. We used the present data to derive guidelines that may allow distance interpretation of other V1-labeled proteins for higher-precision structural modeling

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures

Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) 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 an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs 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 liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context