Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

Abstract

Most proteins in cell and tissue lysates are soluble. Here, we show that many of these proteins, including several that are implicated in neurodegenerative diseases, are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. We identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by doublestranded regions being particularly efficient in this role, revealing an apparent one-to-one protein-nucleic acid stoichiometry. The relationship of these findings to pathological protein aggregation is suggested by our discovery that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble. KEYWORDSmotor neurone disease / protein precipitation / ribonuclease / neurofilament / phase-transition

G‐Quadruplexes act as sequence‐dependent protein chaperones

“…One major barrier existed to using wtGFP to examine chaperone nucleic acid activity. Both in our hands (Fig EV5) and others (Aarum et al , ), bulk nucleic acids have little to no effect on GFP folding or aggregation. This lack of effect likely stems from electrostatic inhibition, as wtGFP is quite acidic (pI = 5.67), and would be repelled by the negative charge of the nucleic acids’ phosphate backbone.…”

RNA modulates physiological and neuropathological protein phase transitions

“…The influence of secondary structure is highlighted by the capacity of short synthetic oligonucleotides that solubilize protein aggregation induced by RNA digestion in whole cell lysate. The most effective solubilizing molecules contain single-stranded motifs, including loops or bulges, interspersed by double stranded regions (Aarum et al, 2020). Similar results have been demonstrated following RBP precipitation via biotinylated isoxazole (b-isox) treatment in HeLa cell lysate, where mass spectrometry revealed preferential antagonization of protein aggregation following incubation with highly structured RNAs, whereas low-structure RNAs produced results similar to background controls (Sanchez de Groot et al, 2019).…”

“…Other disease-associated proteins not typically considered RBPs, such as Tau and Huntingtin, also became aggregated upon RNase digestion, suggesting that RNA:protein interactions contribute to aberrant phase transitions and the resulting protein depositions found in a variety of neurodegenerative disorders. Another key observation of this study was over-representation of proteins associated with the Gene Ontology (GO) terms of ''RNA binding'' and ''translation initiation'' that became aggregated upon RNase treatment, which mirrors the major protein composition of stress granules determined by a similar proteomics analysis (Aarum et al, 2020;Markmiller et al, 2018). These results are especially interesting in light of recent work suggesting a protective role of stress granules in maintenance of TDP-43 and FUS solubility during periods of cellular stress (Gasset-Rosa et al, 2019;Hans et al, 2020;Mann et al, 2019;McGurk et al, 2018;Shelkovnikova et al, 2013).…”

“…Recent proteomics analysis of cell and tissue lysates subjected to RNase treatment additionally demonstrated that this phenomenon is not restricted to a small subset of diseaselinked RBPs because more than 1,300 individual proteins become insoluble following cellular RNA degradation (Aarum et al, 2020). Other disease-associated proteins not typically considered RBPs, such as Tau and Huntingtin, also became aggregated upon RNase digestion, suggesting that RNA:protein interactions contribute to aberrant phase transitions and the resulting protein depositions found in a variety of neurodegenerative disorders.…”