RESUMO
Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.
Assuntos
Crioprotetores/química , Hidrogéis/química , Proteína FUS de Ligação a RNA/química , Sefarose/química , Materiais Biomiméticos/química , Clonagem Molecular , Citoesqueleto/química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Protein-RNA complex formation is at the center of RNA metabolism and leads to the modulation of protein and RNA functions. We propose here a step-by-step guide to investigate these interactions including the identification of the protein and RNA parts involved in complex formation, the determination of the affinity of the complex and the characterization of the protein-RNA interface at amino acid and nucleotide level. Moreover, we briefly review the methods that are the most often used to obtain this information using primarily examples from our lab and finally mention what we perceive as the next challenges in the field.
Assuntos
RNA/genética , Aminoácidos , ProteínasRESUMO
The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.