Biology subjects

Murthy, A. C.

Publications and source records attributed to Murthy, A. C..

6 recordsLinked to original sources

bioRxiv2023

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the additive effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

cell biology↗

bioRxiv2023

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

biochemistry↗

bioRxiv2022

Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

1,6-hexanediol disrupts many phase-separated condensates in cells and in test tubes. In this study, we use a combination of microscopy, nuclear magnetic resonance (NMR) spectroscopy, molecular simulation, and biochemical assays to probe how alkanediols suppress phase separation and why certain isomers are more effective. Alkanediols of different lengths and configurations are all capable of disrupting phase separation of the RNA-binding protein Fused in Sarcoma (FUS), though potency varies depending on both geometry and hydrophobicity, which we measure directly. Alkanediols induce a shared pattern of changes to the protein chemical environment though to differing extents. Consistent with the view that alkanediols disrupt phase separation driven by hydrophobic groups, they decrease the thermal stability of a model globular protein. Conversely, 1,6-hexanediol does not disrupt charge-mediated phase separation, such as FUS RGG-RNA and poly-lysine/poly-aspartic acid condensates. All-atom simulations show that hydroxyl groups in alkanediols mediate interaction with protein backbone and polar amino acid side chains, while the aliphatic chain allows contact with hydrophobic and aromatic residues, providing a molecular picture of how amphiphilic interactions disrupt FUS phase separation.

biochemistry↗

bioRxiv2020

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

biochemistry↗

bioRxiv2019

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

Neurodegenerave diseases such as amyotrophic lateral sclerosis (ALS) are oten associated with mutaons in proteins that are associated with stress granules. Stress granules are condensates formed by liquid-liquid phase separaon which, when aberrant, can lead to altered condensaon behaviours and disease phenotypes. Here, we identified lipoamide, a small molecule which specifically prevents cytoplasmic condensaon of stress granule proteins. Thermal proteome profiling showed that lipoamide preferentially stabilises intrinsically disordered domain-containing proteins. These include SRSF1 and SFPQ, stress granule proteins necessary for lipoamide activity. The redox state of SFPQ correlates with its condensate-dissolving behaviour, in concordance with the importance of the dithiolane ring for lipoamide activity. In animals, lipoamide ameliorates aging-associated aggregaon of a stress granule reporter, improves neuronal morphology, and recovers motor defects caused by expression of ALS-associated FUS and TDP-43 mutants. In conclusion, lipoamide is a well-tolerated small molecule modulator of stress granule condensaon and dissection of its molecular mechanism identified a cellular pathway for redox regulaon of stress granule formaon.

cell biology↗

bioRxiv2018

The FXR2P low complexity domain drives assembly of multiple fibril types with differing ribosome association in neurons

RNA binding proteins (RBPs) typically function in higher order assemblages to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of Fragile X granules, which associate with axonal mRNA and ribosomes in the intact brain. Here we performed an unbiased EGFP insertional mutagenesis screen to probe for FXR2P domains important for assembly into higher order structural states in neurons. Fifteen of the 18 unique in-frame FXR2PEGFP fusions tested formed cytosolic granules. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced formation of distinct FXR2PEGFP fibrils (A and B) that were found in isolation or assembled into highly ordered bundles. Type A and B complexes exhibited different developmental timelines, ultrastructure and ribosome association with ribosomes absent from bundled Type B fibrils. The formation of both fibril types was dependent on an intact RNA binding domain. We conclude that formation of these higher order FXR2P assemblages with alternative structural and compositional states in neurons requires collaboration between the LC and RNA binding domains.\n\nSummary StatementThe Fragile X protein FXR2P forms multiple types of fibrillar assemblages with differential ribosome associations in neurons through cooperation between its RNA binding and LC domains.

neuroscience↗