06 Dec 2013

In times of stress, cells shunt many messenger RNAs into translationally silent stress granules to keep metabolic demands at a minimum. Researchers thought that mutations in FUS and other proteins linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) elicited the same kind of response, because the proteins end up in similar-looking granules. Now, a study in the December 2 Journal of Cell Biology online shows that FUS-containing RNA granules pump out protein. FUS was essential for the translation of mRNAs in granules located in cellular protrusions, suggesting that loss of that translation might damage neurons in “FUSopathies,” according to the research. The work implies that not all RNA granules are the same. Researchers may have to rethink calling RNA-protein globs bona fide, translationally silent stress granules, said Daryl Bosco of the University of Massachusetts Medical School in Worcester, who was not involved in the study.

First author Kyota Yasuda and senior author Stavroula Mili at the National Cancer Institute in Bethesda, Maryland, research RNA-protein complexes found in cellular protrusions such as axon growth cones. These complexes are characterized by the presence of the protein adenomatous polyposis coli (APC), and include several mRNAs (Mili et al., 2008). None of these RNAs are directly related to any disease, Mili told Alzforum. In the study, Yasuda and colleagues used several mRNA members of APC-ribonucleoprotein complexes as convenient markers for the structures:

• Plakopilin 4 (Pkp4), thought to be involved in organizing cell-cell junctions.
• Rab13, which participates in neurite outgrowth.
• KN motif and ankyrin repeat domains 2 (Kank2). Kank proteins regulate actin polymerization and cell movement.
• Discoidin domain receptor tyrosine kinase 2 (Ddr2), an enzyme that regulates cell migration and adhesion to the extracellular matrix.
• Human immunodeficiency virus-1 Tat specific factor 1 (TAT-SF1), a transcription factor that may also participate in RNA splicing.

“Wherever you have protrusions, you have localization of this group of RNAs,” Mili said. These RNAs and others undergo translation in the APC complexes, though how important that translation is to the overall cellular levels of the individual proteins likely varies, since they may be translated within the cell interior as well. For proteins that are already abundant, the APC translation might add little, but it could make a big difference for low-level proteins, Mili said.

How did Mili and Yasuda get from these protrusion granules to ALS-linked FUS? To understand the APC complexes better, they used mass spectrometry to identify proteins that co-immunoprecipitated with APC from mouse fibroblasts. This led them to FUS, a mainly nuclear protein that translocates to the cytoplasm in disease states. “Most of the work on [normal FUS] has overwhelmingly focused on it as a nuclear actor,” commented Benjamin Wolozin of Boston University, who was not part of the study (see Sep 2013 news story). “Here, they are showing a physiological condition where you have FUS out in the cytoplasm.” Mili noted their finding in no way diminishes the importance of nuclear FUS, which makes up the majority of the protein.

To find out what FUS does in APC complexes, Yasuda and colleagues knocked down its expression in the fibroblasts. All of the typical APC complex RNAs still localized to cellular protrusions. However, Kank2 levels fell, indicating that FUS promotes translation of at least some of the APC mRNAs. Wolozin found this surprising, because RNA-binding proteins such as FUS typically silence translation, rather than turning it up.

Next, the researchers studied the effects of FUS mutations associated with ALS. Overexpressing these mutants redistributed the APC-RNA granules, moving them from protrusions to the cellular interior. FUS, APC proteins, and Ddr2 and Pkp4 mRNA accumulated in these interior granules. The granules also contained the stress granule marker T-cell intracellular antigen (TIA-1).

Because researchers who have studied these pathological FUS granules identified them as stress granules (see Alzforum webinar; Jul 2010 news story; Bosco et al., 2010), Mili and colleagues expected that they would be translationally silent. Not so—Kank2, Pkp4, and Ddr2 were produced at normal levels and the FUS-containing granules co-localized with a puromycin label for newly synthesized proteins (see image below). “This goes against the dogma in the field,” commented Bosco. However, Wolozin said he was not entirely surprised. Though scientists typically describe stress granules as translationally silent, he said, their true biology is more complicated and translation may occur in some cases.

Indeed, what this study suggests is that the FUS-containing granules are not traditional stress granules at all, both Mili and Bosco said. “I think we are going to start to appreciate that the properties and functions of these granules depend on the conditions under which they are formed,” Bosco said. For example, cellular stress such as heat shock might lead to a different type of RNA granule than overexpression of a disease-linked protein such as FUS. The criteria to identify a stress granule rely on markers like TIA-1, she noted, not on functional assays. “This paper might cause us to stop and rethink our criteria for saying something is a stress granule, versus some other type of RNA granule,” she said.

Proper characterization matters if one is to understand disease pathology. When examining hippocampal tissue from people who died of FTD, Yasuda saw abnormal granules containing both FUS and APC.

Mili and colleagues are now comparing FUS-containing granules to traditional stress granules. They are also looking at how mislocalization of FUS might affect neurons, since the current study mainly addressed fibroblasts.

Might other ALS and FTD genes contribute to translation in RNA granules, as well? Mili will compare FUS granules to TDP-43 granules. Another gene to consider is repeat-expanded C9ORF72, which was shown earlier this year to form translated dipeptide aggregates as well as mRNA foci (see Feb 2013 news story news story). Researchers have not yet reported where the repeats undergo translation. The RNA and dipeptides rarely coincide; typically a cell contains one or the other but not both. That has led some scientists to suggest that C9ORF72 mRNAs in foci are sequestered away from the translation machinery (see Nov 13 news story).—Amber Dance

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References

News Citations

1. Paper Alert: FUS a Fixer of Damaged DNA 19 Sep 2013
2. Going Nuclear: First Function for FUS Mutants 8 Jul 2010
3. RNA Twist: C9ORF72 Intron Expansion Makes Aggregating Protein 8 Feb 2013
4. Second Study Sees Intron in FTLD Gene Translated 15 Feb 2013
5. Sense, Antisense: C9ORF72 Makes Both Forms of RNA, Peptides 1 Nov 2013

Webinar Citations

1. Stress Granules and Neurodegenerative Disease—What’s the Scoop? 17 Feb 2011

Paper Citations

1. Mili S, Moissoglu K, Macara IG. Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions. Nature. 2008 May 1;453(7191):115-9. PubMed.
2. Bosco DA, Lemay N, Ko HK, Zhou H, Burke C, Kwiatkowski TJ Jr, Sapp P, McKenna-Yasek D, Brown RH Jr, Hayward LJ. Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Hum Mol Genet. 2010 Nov 1;19(21):4160-75. Epub 2010 Aug 10 PubMed.

Further Reading

Papers

1. Bentmann E, Neumann M, Tahirovic S, Rodde R, Dormann D, Haass C. Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43). J Biol Chem. 2012 Jun 29;287(27):23079-94. PubMed.
2. Wolozin B. Regulated protein aggregation: stress granules and neurodegeneration. Mol Neurodegener. 2012;7:56. PubMed.
3. Anderson P, Kedersha N. Stress granules: the Tao of RNA triage. Trends Biochem Sci. 2008 Mar;33(3):141-50. PubMed.
4. Gal J, Zhang J, Kwinter DM, Zhai J, Jia H, Jia J, Zhu H. Nuclear localization sequence of FUS and induction of stress granules by ALS mutants. Neurobiol Aging. 2011 Dec;32(12):2323.e27-40. PubMed.
5. Buchan JR, Parker R. Eukaryotic stress granules: the ins and outs of translation. Mol Cell. 2009 Dec 25;36(6):932-41. PubMed.
6. Weil TT, Parton RM, Herpers B, Soetaert J, Veenendaal T, Xanthakis D, Dobbie IM, Halstead JM, Hayashi R, Rabouille C, Davis I. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat Cell Biol. 2012 Dec;14(12):1305-13. Epub 2012 Nov 25 PubMed.

News

1. Is FUS to Blame for Neurons Vulnerable to FTLD and ALS? 16 Aug 2013
2. Disease Mutations Zip Lock Stress Granules in Proteinopathy, ALS 7 Mar 2013
3. Friends of FUS: Protein's Many RNA Buddies Point to Disease 4 Oct 2012
4. Paper Alert: Stress Granules Get Tangled Up in Tau 13 Jun 2012
5. DC: ALS Treatment Possibilities Presented at SfN, Satellite 30 Nov 2011
6. London, Ontario: The Fuss About FUS at ALS Meeting 6 Jul 2009