RNA Metabolism in ND 2015: Does C9ORF72 Repeat RNA Promote Protein Phase Transitions?

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Over the past couple of months, scientists have learned that RNA-binding proteins such as FUS and hnRNPA1 undergo phase transitions, separating themselves and their associated nucleic acids and protein partners into liquid-phase-like organelles that are distinct from the surrounding cytoplasm or nucleoplasm (see Alzforum Webinar). At the third biennial RNA Metabolism in Neurological Disease meeting, held in Chicago October 15-16, researchers from Brigham and Women’s Hospital in Boston offered preliminary data indicating that RNAs themselves might seed formation of these liquid organelles, too. It looks as if RNAs might manage the same trick that previously only proteins were thought to perform, said Marta Fay, who presented the work in a poster. The meeting was a satellite to the Society for Neuroscience meeting.

Pernicious seed. C9ORF72 RNAs form G-quadruplexes that attract RNA-binding proteins, leading to large accumulations that might take the form of aggregates or hydrogels. [Courtesy of Marta Fay, Brigham and Women’s Hospital.]

Pernicious seed. C9ORF72 RNAs form G-quadruplexes that attract RNA-binding proteins, leading to large accumulations that might take the form of aggregates or hydrogels. [Courtesy of Marta Fay, Brigham and Women’s Hospital.]

Fay, who works in the laboratory of Paul Anderson, and Brigham collaborator Pavel Ivanov studied RNA from C9ORF72, a gene implicated in amyotrophic lateral sclerosis and frontotemporal dementia. Carriers of the expanded gene often express hundreds or thousands of GGGGCC repeats in their RNA, as well as CCCCGG repeats made from transcription in the antisense direction (see Sep 2011 news; Jan 2013 conference news; Nov 2013 news). Scientists suspect the repetitive RNAs, which assemble into foci, could be toxic. Might they cause problems by inducing liquid- or solid-phase transitions in the cell?

If so, Fay would expect to see the C9ORF72 RNAs associating with components of liquid organelles such as stress granules. She incubated the repeat RNAs with lysates from mouse brain or cultured U2OS cells, a commonly used human osteosarcoma line in which stress granules have been studied in the past. Once Fay added the C9ORF RNAs, she observed the formation of precipitates. Mass spectrometry identified 187 molecules in them, including known stress granule constituents such as G3BP1, eIF3b, mRNAs, and ribosome subunits. Other precipitated proteins are not typically found in stress granules, but some normally make up the related P bodies, suggesting the C9ORF72 RNAs associated with the members of multiple types of RNA granules.

As a control, Fay compared her precipitation process to that induced by biotinylated isoxazole (b-isox), a chemical known to shepherd RNA granule components into a hydrogel (Kato et al., 2012). C9ORF72 RNA acted like b-isox, precipitating the RNA-binding proteins in a temperature- and concentration-dependent manner.

These precipitates only formed when Fay added GGGGCC. The antisense CCCCGG RNA yielded no aggregation. The sense repeats are known to assemble into structures called G-quadruplexes, where G-rich sequences within a single RNA strand line up like the four sides of a building (see image above). Fay thought this might explain why only the sense sequence precipitates RNA granule components. It did. When Fay added drugs that promoted G-quadruplex formation, she saw more precipitates. When she applied treatments that dissolved the quadruplexes, she obtained less.

Finally, Fay tested her ideas in cells, again from the U2OS line. Expressing the repeat RNA in those cells induced formation of stress granules, and the longer the repeats, the more granules appeared. She proposed that the extensive C9ORF72 repeats might over-stabilize granules, pushing the proteins therein toward pathological fibrilization (see image above).

Researchers are not sure yet precisely what these precipitates are, and if they are liquid, gel-like, or solid in nature. Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, a meeting co-organizer and panelist at the Alzforum Webinar, thought Fay’s results were not necessarily indicative of phase separation. Ivanov said he and his colleagues would collaborate with biophysicists to solve the question.

Supporting Fay’s findings, other scientists have discovered that RNAs are not merely caught up in granule organelles, but actively participate in the phase transition. “RNA can impact both the assembly of droplets and their properties,” commented Cliff Brangwynne of Princeton University, a pioneer of the field of protein phase transition. Brangwynne did not attend the Chicago meeting but will lead off today’s Webinar. “RNA can also influence the viscoelastic properties of the droplets,” he told Alzforum (Berry et al., 2015; Elbaum-Garfinkle et al., 2015; Zhang et al., 2015).


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c9orf72 conf-rna-metabolism-in-neurological-disease-conference disease-als disease-ftd FUS tdp-43 topic-preclinical
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