Does C9ORF72 Repeat RNA Promote Protein Phase Transitions?

Series - 10th Brain Research Conference: RNA Metabolism in Neurological Disease:

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 today’s 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.]

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).—Amber Dance

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References

Webinar Citations

  1. Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?

News Citations

  1. Corrupt Code: DNA Repeats Are Common Cause for ALS and FTD
  2. Chicago—RNA Inclusions Offer Therapeutic Target in ALS
  3. Sense, Antisense: C9ORF72 Makes Both Forms of RNA, Peptides

Paper Citations

  1. Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Longgood J, Pei J, Grishin NV, Frantz DE, Schneider JW, Chen S, Li L, Sawaya MR, Eisenberg D, Tycko R, McKnight SL. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell. 2012 May 11;149(4):753-67. PubMed.
  2. Berry J, Weber SC, Vaidya N, Haataja M, Brangwynne CP. RNA transcription modulates phase transition-driven nuclear body assembly. Proc Natl Acad Sci U S A. 2015 Sep 22;112(38):E5237-45. Epub 2015 Sep 8 PubMed.
  3. Elbaum-Garfinkle S, Kim Y, Szczepaniak K, Chen CC, Eckmann CR, Myong S, Brangwynne CP. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc Natl Acad Sci U S A. 2015 Jun 9;112(23):7189-94. Epub 2015 May 26 PubMed.
  4. Zhang H, Elbaum-Garfinkle S, Langdon EM, Taylor N, Occhipinti P, Bridges AA, Brangwynne CP, Gladfelter AS. RNA Controls PolyQ Protein Phase Transitions. Mol Cell. 2015 Oct 15;60(2):220-30. PubMed.

Further Reading

Papers

  1. Weber SC, Brangwynne CP. Getting RNA and protein in phase. Cell. 2012 Jun 8;149(6):1188-91. PubMed.
  2. Becker LA, Gitler AD. It's all starting to come together. Elife. 2015 Aug 5;4 PubMed.
  3. Lee CF, Brangwynne CP, Gharakhani J, Hyman AA, Jülicher F. Spatial organization of the cell cytoplasm by position-dependent phase separation. Phys Rev Lett. 2013 Aug 23;111(8):088101. Epub 2013 Aug 20 PubMed.
  4. Kedersha N, Ivanov P, Anderson P. Stress granules and cell signaling: more than just a passing phase?. Trends Biochem Sci. 2013 Oct;38(10):494-506. Epub 2013 Sep 10 PubMed.
  5. Li P, Banjade S, Cheng HC, Kim S, Chen B, Guo L, Llaguno M, Hollingsworth JV, King DS, Banani SF, Russo PS, Jiang QX, Nixon BT, Rosen MK. Phase transitions in the assembly of multivalent signalling proteins. Nature. 2012 Mar 7;483(7389):336-40. PubMed.
  6. Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F, Hyman AA. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 2009 Jun 26;324(5935):1729-32. Epub 2009 May 21 PubMed.

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