A yeast functional screen predicts new candidate ALS disease genes

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregationprone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. I n the future, personalized genome sequencing will become routine, empowering us to define the genetic basis of many human diseases. Currently, however, complete genome sequencing for individuals to discover rare pathogenic mutations is still too costly and time consuming. Thus, more creative approaches are needed to accelerate the discovery of disease genes. Moreover, even once genes are revealed, the need for innovative approaches to elucidate causality remains critical.ALS, also known as Lou Gehrig's disease, is a devastating adultonset neurodegenerative disease that attacks upper and lower motor neurons (1). A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2-5 y of disease onset. ALS is mostly sporadic, but ∼10% of cases are familial. Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN, VCP, and others (2). Two of these genes, TARDBP (TDP-43) and FUS/TLS (FUS) are notable because they encode related RNA-binding proteins that harbor a prion-like domain (3-6). Moreover, both of these proteins have been identified as components of pathological inclusions in neurons of patients with ALS (7-9). Indeed, an emerging concept suggested by the association of FUS and TDP-43 to ALS is that defects in RNA metabolism might contribute to disease pathogenesis. These observations suggested an intriguing possibility: Could TDP-43 and FUS be just the tip of an iceberg? In other words, could other human RNA-binding proteins with properties similar to th...

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

“…Its 580 RNDYRNDQR 588 segment probably forms a similar polarized central helix and its C-terminal conserved PY motif is most likely also structurally conserved. EWS and TAF15 were recently suggested to also be mutated in some rare ALS cases (25)(26)(27)(28). The few disease mutation sites found thus far are located in the RGG regions that are N-terminal of the PY-NLSs.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…The RNA-binding ability of TDP43, hnRNP A1, and hnRNP A2/B1 may be key to the propagation of neurodegeneration, as RNA is a potent and necessary cofactor for the conversion of the mammalian prion protein PrP C into its pathogenic isoform, PrP Sc [128]. Confirming the connection between neurodegeneration and prion-like behavior of RNA-binding proteins, algorithms designed to identify proteins harboring prion-like domains revealed an enrichment of RNA binding proteins [28,31], many of which have since been implicated in ALS and FTLD pathogenesis [30,129].…”

“…Many of the factors associated with TDP43 and FUS are components of cytoplasmic RNA stress granules [poly(A)-binding protein 1, cytotoxic granule-associated RNA binding protein] or miRNA processing complexes (Drosha, Dicer). In addition, TDP43 binds Matrin 3, VCP, EWSR1, TAF15, and FUS, all of which can cause fALS when mutated [9,15,16,19,30,31].…”

“…TDP43 also shares significant structural and functional homology with fused in sarcoma (FUS), an RNA-binding protein that is part of the FET family of RNA/DNA-binding proteins that includes FUS, Ewing sarcoma breakpoint region 1 (EWSR1), and TATA box binding protein-associated factor 15 (TAF15). Supporting the connection between abnormal RNA processing and motor neuron disease, nonsense or missense mutations in FUS, EWSR1, and TAF15 are associated with fALS [30,31]. Moreover, trinucleotide (CAG) expansion mutations in ATXN2, encoding the RNA-binding protein ataxin-2, cause spinocerebellar ataxia or ALS, depending on the number of CAG repeats [32][33][34][35], and, in some populations, hexanucleotide (G 4 C 2 ) expansion mutations in the noncoding region of the C9orf72 locus account for up to 40 % of fALS [17,18].…”

RNA-binding proteins in neurological diseases

“…A screen in yeast identified 133 human RBPs, that were cytotoxic and formed cytoplasmic aggregates, similar to TDP-43 and FUS [153]. Some of these RBPs contain prion-like sequences.…”