04 Oct 2012

Mutations in two RNA-binding proteins, TDP-43 and FUS, cause amyotrophic lateral sclerosis (ALS). These proteins form aggregates in ALS as well as in frontotemporal dementia (FTD). This suggests RNA dysregulation is part of these diseases, and begs an obvious question: Which RNAs? For TDP-43, several research groups have already identified thousands of RNA targets (see ARF related news story on Sephton et al., 2011, and ARF related news story on Tollervey et al., 2011, and Polymenidou et al., 2011). Two of those teams now show that FUS binds a separate plethora of targets—with a few conspicuous exceptions. Both FUS and TDP-43 appear to stabilize a small group of the same RNAs, including several involved in maintaining neurons. The results may help researchers identify pathological pathways common to both diseases.

Researchers from the University of California, San Diego, laboratories of Don Cleveland and Gene Yeo report that, though FUS and TDP-43 share few RNA targets, they do both promote the expression of neural genes that possess lengthy introns. Published in the September 30 Nature Neuroscience, this work follows another study of FUS targets led by Jernej Ule of the Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge, U.K., and Chris Shaw of King’s College, London. In the August 28 Scientific Reports, a new open-access journal by Nature Publishing Group, the authors describe FUS’ role in mediating alternative splicing, where it prevents target exons from being included in mature mRNAs.

Target Practice
Both the U.S. and U.K. teams used a strategy called crosslinking and immunoprecipitation (CLIP) to identify FUS-RNA interactions. They applied ultraviolet light to crosslink protein-RNA complexes in mouse brain lysates, and then used antibodies to pull down FUS and its partners. Magdalini Polymenidou, Clotilde Lagier-Tourenne, and Kasey Hutt, joint first authors on the Nature paper, found that FUS bound to 5,500 target mRNAs. They repeated the experiment with human cortex and found similar FUS targets. For their part, Ule’s group found that FUS bound to all pre-mRNAs that contain introns in mouse brain samples.

Ule said FUS tagged so many mRNAs because it is fairly indiscriminate in where it binds. Though it preferred GGU-containing motifs, it was not limited to those sequences. TDP-43, in contrast, only tends to latch on to specific, UGUG-rich sites. FUS is also unique among RNA-binding proteins, Ule said, in that it attaches along wide swaths of mRNA sequences.

Not only did FUS bind long stretches of mRNA, but it also appeared bound to unfinished transcripts. That means FUS grabbed onto nascent RNAs as soon as they were transcribed off the DNA. Lagier-Tourenne suggested that this could mean FUS plays a role during ongoing transcription. Supporting this idea, FUS is known to interact with RNA polymerase (Bertolotti et al., 1998). The idea is not unprecedented; another splicing factor, SC35, also regulates transcription (Lin et al., 2008).

While FUS may bind many targets, scientists are most interested in the targets on which it exerts measureable effects. Ule and first author Boris Rogelj, formerly of Shaw’s lab and now an independent researcher at the Josef Stefan Institute in Ljubljana, Slovenia, investigated FUS’ role in RNA splicing. They isolated RNA from the brain tissue of wild-type and FUS knockout mouse embryos. The researchers analyzed these samples with microarrays designed to identify splice site junctions. They found 68 differences in alternative splicing between the samples from FUS-positive and FUS-negative mice. The majority represented exons that are normally spliced out, but were included in mature mRNAs in the FUS knockout brains. Ule concluded that FUS normally must repress the inclusion of these exons during splicing. “The way FUS regulates splicing is very different from any other protein we have checked before,” he noted.

The UCSD researchers focused on how FUS controls the overall expression of different RNAs, and compared its effects to those of TDP-43 from their previous work. They used antisense oligonucleotides to knock down FUS in the brains of adult mice, and then analyzed the RNAs present in the striatum. Of the thousands of mRNAs that FUS binds, it regulated the levels of 610; 112 of those also bound TDP-43. A majority of the 112 were downregulated by one or the other, or both, but 45 required both FUS and TDP-43 for full expression.

The researchers were most interested in these 45 genes. Many contained extra-long introns, on the order of 160 kilobases, and several of these long-intron genes are known to be involved in the biology of the neuron. “It is clear that FUS is necessary to maintain these transcripts,” Lagier-Tourenne said. How it does so is unknown.

RNAs Raise Further Questions
What does this long list of targets tell researchers about ALS and FTD? “I have been wondering for some time whether [TDP-43 and FUS] are on the same pathway, and whether they regulate the same genes,” commented Chantelle Sephton of the University of Texas Southwestern Medical Center in Dallas, who was not involved in either study. “Ideally you would find one RNA target that is affected [by both] … which would make it easy for us to understand how [those RNAs] relate to disease.” Unfortunately, nature made the story more difficult.

Lagier-Tourenne said all the targets TDP-43 and FUS have in common are likely to be involved in ALS and FTD. Because TDP-43 and FUS vacate the nucleus in the neurons of people with those diseases, the RNAs dependent on them presumably suffer misregulation. “This work provides additional, powerful evidence for [loss of function] as a significant part of the disease etiology,” wrote Dagmar Ringe and Gregory Petsko of Brandeis University in an e-mail to Alzforum (see full comment below). However, that does not preclude a gain-of-function mechanism as well.

Which of TDP-43's and FUS’ RNAs will prove key to ALS and FTD? It is not possible yet to say. However, the FUS studies picked out several genes sure to pique the interest of scientists studying neurodegeneration. The MAPT gene encoding tau appears to rely on FUS for proper splicing, as does sortilin, a receptor for the FTD-linked growth factor progranulin. Parkin is one of those long-intron genes; it depends on TDP-43 and FUS for proper expression in human neural progenitors derived from stem cells, the UCSD team reported. And in motor neurons from people who died of ALS, the scientists found that TDP-43 cytoplasmic aggregates were correlated with a reduction in parkin staining by immunofluorescence.

With so many RNAs to choose from, it is too early to start fitting FUS and TDP-43 targets into a specific model for disease, Sephton said. She suggested that researchers should examine the RNA-regulating profiles of disease-linked FUS and TDP-43 mutants to narrow down the list.

“Our current studies are not conclusive on their own,” Ule agreed. “They are setting the stage for the future.” For example, Lagier-Tourenne suggested researchers might use the drop in expression of long-intron genes as a molecular marker for TDP-43 pathology. And if scientists go hunting for other RNA-binding proteins that regulate the same subset of RNAs, those might turn out to be key ALS genes, proposed Aaron Gitler of Stanford University in Palo Alto, California, who was not involved in either study.—Amber Dance

Comments

1. • Dagmar Ringe
     Brandeis University
   • Posted: 04 Oct 2012
   • Paper: Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs.

   This is a very important study. The fact that loss of function may underlie at least some of the toxicity of TDP-43 and FUS/TLS mutations in ALS is not itself surprising—a number of previous observations had suggested this. But this work provides additional, powerful evidence for that as a significant part of the disease etiology. In addition, by identifying specific, common target RNAs for these two proteins, this elegant work offers an explanation for the fact that our own laboratory, together with that of Steve Finkbeiner at UCSF, has found that suppression of FUS/TLS toxicity by specific genes and chemical substances usually is accompanied by suppression of TDP-43 toxicity by these same agents, in both model organisms and neuronal cell cultures. Among the common targets identified in this paper may be those that are responsible for the overlapping disease mechanism that seems to apply in these forms of ALS. It is hard to believe that these results won't eventually contribute to finding a treatment for this terrible disease.

   View all comments by Dagmar Ringe

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References

News Citations

1. San Diego: TDP-43 Targets Loom Large—But Where’s the Bull’s Eye? 25 Nov 2010
2. CLIPs of TDP-43 Provide a Glimpse Into Pathology 9 Mar 2011

Paper Citations

1. Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han Y, Dewey CM, Roth FP, Herz J, Peng J, Moore MJ, Yu G. Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem. 2011 Jan 14;286(2):1204-15. PubMed.
2. Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci. 2011 Apr;14(4):452-8. PubMed.
3. Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. 2011 Apr;14(4):459-68. PubMed.
4. Bertolotti A, Melot T, Acker J, Vigneron M, Delattre O, Tora L. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol Cell Biol. 1998 Mar;18(3):1489-97. PubMed.
5. Lin S, Coutinho-Mansfield G, Wang D, Pandit S, Fu XD. The splicing factor SC35 has an active role in transcriptional elongation. Nat Struct Mol Biol. 2008 Aug;15(8):819-26. PubMed.

Further Reading

Papers

1. Budini M, Baralle FE, Buratti E. Regulation of gene expression by TDP-43 and FUS/TLS in frontotemporal lobar degeneration. Curr Alzheimer Res. 2011 May 1;8(3):237-45. PubMed.
2. Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurol. 2010 Oct;9(10):995-1007. PubMed.
3. Kim SH, Shanware NP, Bowler MJ, Tibbetts RS. Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA. J Biol Chem. 2010 Oct 29;285(44):34097-105. PubMed.
4. van Blitterswijk M, Landers JE. RNA processing pathways in amyotrophic lateral sclerosis. Neurogenetics. 2010 Jul;11(3):275-90. PubMed.
5. Traynor BJ, Singleton AB. What's the FUS!. Lancet Neurol. 2009 May;8(5):418-9. PubMed.
6. Ishigaki S, Masuda A, Fujioka Y, Iguchi Y, Katsuno M, Shibata A, Urano F, Sobue G, Ohno K. Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions. Sci Rep. 2012;2:529. PubMed.

News

1. Could Sortilin Be a Sweet Spot for FTD Therapy? 21 Aug 2012
2. RNA Processors Turn Up in Another Motor Neuron Disease 2 May 2012
3. London, Ontario: The Fuss About FUS at ALS Meeting 6 Jul 2009
4. CLIPs of TDP-43 Provide a Glimpse Into Pathology 9 Mar 2011
5. Yeast Models Say TDP-43 and FUS Are Not Cut From the Same Cloth 10 May 2011
6. Québec: Teasing Out the Function of TDP-43 4 Oct 2010
7. Going Nuclear: First Function for FUS Mutants 8 Jul 2010
8. New Gene for ALS: RNA Regulation May Be Common Culprit 27 Feb 2009
9. Paris: Diabetes, Insulin, and Alzheimer Disease 17 Jun 2009