Genetic interaction of hnRNPA2B1 and DNAJB6 in aDrosophilamodel of multisystem proteinopathy
Songqing LiPeipei ZhangBrian D. FreibaumNam Chul KimRegina-Maria KolaitisAmandine MolliexAnderson KanagarajIchiro YabeMishie TaninoShinya TanakaHidenao SasakiEric D. RossJ. Paul TaylorHong Joo Kim
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Adult-onset inherited myopathies with similar pathological features, including hereditary inclusion body myopathy (hIBM) and limb-girdle muscular dystrophy (LGMD), are a genetically heterogeneous group of muscle diseases. It is unclear whether these inherited myopathies initiated by mutations in distinct classes of genes are etiologically related. Here, we exploit a genetic model system to establish a mechanistic link between diseases caused by mutations in two distinct genes, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila melanogaster homologs of human hnRNPA2B1 and DNAJB6, respectively. We introduced disease-homologous mutations to Hrb98DE, thus capturing mutation-dependent phenotypes in a genetically tractable model system. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins (RBPs), suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover, wild-type, but not disease-associated, mutant forms of MRJ interacted with RBPs after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interactions between disease-linked RBPs and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.Keywords:
Ectopic expression
Mutations in the spineless-aristapedia (ssa) gene of Drosophila melanogaster are pleiotropic, and their classical manifestations include a reduction in size of all bristles (spineless phenotype), transformation of distal parts of antennae into tarsal segments of the mesothoracic leg (aristapedia phenotype), and, in extreme alleles, fusion of tarsal segments on all six legs and the transformed aristaes. We isolated a new allele, which is a severe loss-of-function mutation and, in addition to the above-mentioned features, is characterized by amplification of sex combs on the first leg. This phenotype can be caused by a change in the expression of the Sex combs reduced (Scr) gene of the ANTP-C. Identification of this phenotype, together with observed variations in the extent of the fusion of tarsal segments in the legs of different segments, raised the possibility that ssa interacts with homeotic genes controlling the identity of segments. This possibility was tested in genetical experiments using flies with loss-of-function mutations in several homeotic genes and flies transformed by heat shock-driven homeotic genes. Analysis of adult phenotypes of different ssa alleles in the background of under-, over-, or ectopic expression of some genes of BX-C and ANT-C suggests that the ssa product is required to prevent the effect of the homeotic gene products in the distal segments of the appendages.
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Dysferlin
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To determine the phenotype variability associated with the specific C-terminal M-line titin mutation known to cause autosomal dominant distal myopathy, tibial muscular dystrophy (TMD; MIM 600334), and limb girdle muscular dystrophy 2J (LGMD2J).Three hundred eighty-six individuals were genotyped for the Finnish founder mutation in titin (FINmaj) causing TMD/LGMD2J.Two hundred seven patients were heterozygous for the mutation. Among these patients, 189 (91%) had a more common phenotype compatible with the classic description of TMD. However, 18 (9%) had unusual phenotypes such as proximal leg or posterior lower leg muscle weakness and atrophy even at onset. Four patients were confirmed homozygotes representing the LGMD2J phenotype. These homozygotes were half of the eight LGMD patients previously described in the original large consanguineous kindred.Large variability of phenotypic expression caused by just one mutation, the Finnish FINmaj, suggests that no certain phenotype of myopathy/dystrophy can be excluded from being caused by mutated titin. Yet unknown homozygous or compound heterozygous titin mutations without phenotype in the heterozygote carriers may be responsible for undetermined recessive MD and LGMD.
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Heterozygote advantage
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White (mutation)
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Abstract The neurogenic genes of Drosophila melanogaster are involved in the decision of ectodermal cells to take on a neural or an epidermal fate. We present evidence in support of the notion that six of the neurogenic genes are functionally related. We studied the phenotype of embryos lacking one of the neurogenic genes in the presence of an increased dosage of the wild-type allele of another neurogenic gene. Our analysis also included the Hairless locus, whose function is related to that of the neurogenic genes, as well as to many other genes. The effects observed were asymmetric in that triploidy for a given gene modified the phenotype of loss of the function of another gene, but triploidy of the latter gene did not modify the phenotype of loss of the function of the former gene. These asymmetries allowed us to establish a polarity of gene interactions, as well as to order the genes according to the assumed ability of some of them to modify the activity of others. In this sequence, almondex is the first link and Enhancer of split the last one. Our evidence suggests that the function of big brain is independent of the function of the other six. The consequences of this arrangement for the commitment of ectodermal cells are discussed.
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We studied facultative dominant lethal mutations obtained earlier in Drosophila melanogaster. In some genotypes, these mutations were expressed as lethals, but in other genotypes they lacked this expression. The mutations were maintained in the following cultures: (1) females Muller-5 heterozygous for the mutation; (2) males crossed to attached-X females; and females and males homozygous for the mutation. During culturing, many mutations were found to give rise to phenotypically abnormal progeny. Generally, these abnormalities were morphoses involving various body parts; they were mostly asymmetric and non-heritable. Maternal and paternal effects in the formation of morphoses were observed. In four cases, dimorphic mutations were recorded: a female homozygous for the mutation had mutant phenotype whereas its male counterpart was phenotypically normal. The mutations were recessive with regard to the norm. New phenotypes behaving as mutations with incomplete penetrance arose during culturing. In cultures of mutant homozygotes phenocopies would appear en masse; they would persist for one or two generations and disappear. One wave of phenocopies succeeded another. Visible phenotypes appeared, which further behaved as ordinary recessive mutations. We concluded that these visible manifestations are characteristic for regulatory mutations controlling ontogeny. Their appearance is explained by the activation of new regulatory scenarios caused by blocking standard regulatory pathways.
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An analysis of revertants of missense mutants in phage P22 has shown: (i) New temperature-sensitive (TS) and cold-sensitive (CS) phenotypes are often acquired concomitant with reversion. (ii) In many cases, these new phenotypes are due to second-site mutations (suppressors) that correct the original defect. (iii) Sometimes the suppressor mutation is not in the same gene as the original mutation. (iv) Extragenic suppressors are almost always in genes whose products are known to interact physically with the original gene products. (v) The suppressor mutations typically retain their TS or CS phenotypes when crossed into wild-type genetic backgrounds. (vi) Some TS and CS mutants derived by reversion can themselves be reverted to produce additional mutations. We have shown that genetic reversion of missense mutants can be of value in producing new temperature-sensitive and cold-sensitive mutations affecting related functions. We suggest that our approach can be extended to organisms with large genomes.
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Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.
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The analysis of the X-linked wisertsl (1-21.7, 7E) mutation in Drosophila melanogaster has shown that responsible for the scalloped phenotype and the temperature sensitivity is the CG32711 gene, which we name wiser (wings scalloped-eyes rough). The gene wiser is essential for Drosophila development. The wisertsl mutation is mapped at the 5′ regulatory region of the gene CG32711. The wiserPL26 lethal mutation is mapped in the same region. Using these two mutations and a UAS-wiser transgene we have shown that: a) The wisertsl and wiserPL26 mutations increase the wing scalloping (phenotype) of the mutations Beadex1 and Serrate1. The genes Beadex and Serrate are implicated in the activation of Notch signaling pathway along the dorsal-ventral axis of the wing. This observation indicates that the wiser gene is involved in determination of dorsal-ventral axis. b) The wisertsl mutation in homozygous condition reduces substantially the expression of fringe-lacZ, m8-lacZ, wingless-lacZ, vestigial-lacZ and Distalless-lacZ transgenes, alters the expression pattern of mβ-lacZ and does not affect the expression of apterous-lacZ transgene in the wing imaginal disc. This indicates that the expression of fringe (a modifier of Notch receptor) is regulated by wiser too. c) Ectopic expression of UAS-wiser by the ap-Gal4 driver partially rescues apterous- but not Serrate1 phenotype. d) Ectopic expression of UAS-wiser by the dpp-Gal4 driver affects the expression of wingless and does not affects the expression of apterous, fringe, mβ, m8, vestigial and Distalless in the wing imaginal disc (revealed by the corresponding -lacZ strains). e) Induction of somatic clones with the FRT/FLP system in wiserPL26/+ mutants led to mitotic +/+ and wiserPL26/wiserPL26 clones of different sizes. The first clones were much larger than the second ones in the territory of wing pouch. Adult females with scalloped wings were also produced. These results indicate that the wiser gene is involved in cell proliferation. All the above findings suggest that the wiser gene is essential for wing development and cell proliferation.
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