20 research outputs found
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Prion-like domain mutations in hnRNPs cause multisystem proteinopathy and ALS
Summary Algorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone
Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas
Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors
Characterization of the Novel Telomere Associated Protein: hSnm1B
<p>Telomeres are the ends of chromosomes which are composed of repetitive DNA sequence and telomere associated proteins. In C. elegans, the protein F39H2.5 was found to associate with the telomere, regulating both telomere length and genomic integrity. F39H2.5 is a member of the β-CASP family of proteins that are known to possess nuclease activity on DNA substrates. I thus sought to address whether any of the human β-CASP family proteins associated with telomeres. Here I show that hSnm1B localized to the telomere indirectly, via interaction with the double-stranded telomere binding protein TRF2. The terminal 37 amino acids of hSnm1B are necessary and sufficient for binding TRF2, and moreover that binding to TRF2 stabilized hSnm1B protein by preventing ubiquitination. In the absence of exogenous TRF2 this domain acted as a degron, promoting protein instability. I thus termed the domain the Protection And INstability (PAIN) domain. I hypothesize that TRF2 binding ensures that hSnm1B will only accumulate at telomeres by preventing the degradation of hSnm1B. However, hSnm1B stability appears to be further regulated, as telomere specific DNA damage stabilized hSnm1B independent of the PAIN domain. Thus, it appears that the telomere associated protein, hSnm1B, is regulated by protein stability in a manner that is both dependent and independent of the PAIN domain.</p>Dissertatio
Distinct Functions of POT1 at Telomeres â–¿ â€
The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions—ssDNA, TPP1, or TRF2 binding—was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres
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Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity.
Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS
The Protein hSnm1B Is Stabilized When Bound to the Telomere-binding Protein TRF2*
hSnm1B is member of the SNM family of exonucleases involved in DNA
processing and is known to be localized to telomeres via binding to the
telomere-binding protein TRF2. Here we demonstrate that the C terminus of
hSnm1B facilitates the concentration of hSnm1B on telomeres by promoting
ubiquitin-mediated degradation of hSnm1B that is not localized to telomeres,
as well as by blocking protein degradation and fostering localization to
telomeres via binding of TRF2. Finally, a mutant of hSnm1B stabilized
independently of exogenous TRF2-induced cell death. Taken together, we
speculate that sequestering hSnm1B at telomeres by a combination of
stabilizing the protein when bound to telomeres and degrading it when not
bound to telomeres may be a means to prevent potentially lethal effects of
unregulated hSnm1B activity
Axonal Transport of TDP-43 mRNA Granules Is Impaired by ALS-Causing Mutations
SummaryThe RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43