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  • 1
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    Splicing factor DHX15 affects tp53 and mdm2 expression via alternate splicing and promoter usage
    Oxford University Press (OUP) ; 2019
    In:  Human Molecular Genetics Vol. 28, No. 24 ( 2019-12-15), p. 4173-4185
    Details
    In: Human Molecular Genetics, Oxford University Press (OUP), Vol. 28, No. 24 ( 2019-12-15), p. 4173-4185
    Abstract: DHX15, a DEAH box containing RNA helicase, is a splicing factor required for the last step of splicing. Recent studies identified a recurrent mutational hotspot, R222G, in DHX15 in ∼ 6% of acute myeloid leukemia (AML) patients that carry the fusion protein RUNX1-RUNX1T1 produced by t (8;21) (q22;q22). Studies using yeast mutants showed that substitution of G for the residue equivalent to R222 leads to loss of its helicase function, suggesting that it is a loss-of-function mutation. To elucidate the role of DHX15 during development, we established the first vertebrate knockout model with CRISPR/Cas9 in zebrafish. Our data showed that dhx15 expression is enriched in the brain, eyes, pectoral fin primordia, liver and intestinal bulb during embryonic development. Dhx15 deficiency leads to pleiotropic morphological phenotypes in homozygous mutant embryos starting at 3 days post fertilization (dpf) that result in lethality by 7 dpf, revealing an essential role during embryonic development. RNA-seq analysis suggested important roles of Dhx15 in chromatin and nucleosome assembly and regulation of the Mdm2-p53 pathway. Interestingly, exons corresponding to the alternate transcriptional start sites for tp53 and mdm2 were preferentially expressed in the mutant embryos, leading to significant upregulation of their alternate isoforms, Δ113p53 (orthologous to Δ133p53 isoform in human) and mdm2-P2 (isoform using distal promoter P2), respectively. We speculate that these alterations in the Mdm2-p53 pathway contribute to the development of AML in patients with t(8;21) and somatically mutated DHX15.
    Type of Medium: Online Resource
    ISSN: 0964-6906 , 1460-2083
    URL: Article
    DOI: 10.1093/hmg/ddz261
    RVK:
    XA 10000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
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  • 2
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    A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
    Springer Science and Business Media LLC ; 2016
    In:  Nature Protocols Vol. 11, No. 12 ( 2016-12), p. 2357-2375
    Details
    In: Nature Protocols, Springer Science and Business Media LLC, Vol. 11, No. 12 ( 2016-12), p. 2357-2375
    Type of Medium: Online Resource
    ISSN: 1754-2189 , 1750-2799
    URL: Article
    DOI: 10.1038/nprot.2016.141
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
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    detail.hit.zdb_id: 2732852-1
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  • 3
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    Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues
    Springer Science and Business Media LLC ; 2018
    In:  npj Regenerative Medicine Vol. 3, No. 1 ( 2018-06-04)
    Details
    In: npj Regenerative Medicine, Springer Science and Business Media LLC, Vol. 3, No. 1 ( 2018-06-04)
    Abstract: Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.
    Type of Medium: Online Resource
    ISSN: 2057-3995
    URL: Article
    DOI: 10.1038/s41536-018-0050-7
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2018
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  • 4
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    High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
    Cold Spring Harbor Laboratory ; 2015
    In:  Genome Research Vol. 25, No. 7 ( 2015-07), p. 1030-1042
    Details
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 25, No. 7 ( 2015-07), p. 1030-1042
    Abstract: The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F 1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    URL: Article
    DOI: 10.1101/gr.186379.114
    RVK:
    XA 10000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2015
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  • 5
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    Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish
    MDPI AG ; 2022
    In:  International Journal of Molecular Sciences Vol. 23, No. 18 ( 2022-09-14), p. 10668-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 23, No. 18 ( 2022-09-14), p. 10668-
    Abstract: ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms231810668
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
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  • 6
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    Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors
    BMJ ; 2018
    In:  Annals of the Rheumatic Diseases Vol. 77, No. 4 ( 2018-04), p. 612-619
    Details
    In: Annals of the Rheumatic Diseases, BMJ, Vol. 77, No. 4 ( 2018-04), p. 612-619
    Abstract: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1 , a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. Methods We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients’ primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). Results We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1β were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients’ fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. Conclusions Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.
    Type of Medium: Online Resource
    ISSN: 0003-4967 , 1468-2060
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.1136/annrheumdis-2017-212401
    DOI: 10.1136/annrheumdis-2017-212401.supp1
    DOI: 10.1136/annrheumdis-2017-212401.supp2
    RVK:
    XA 10000
    Language: English
    Publisher: BMJ
    Publication Date: 2018
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  • 7
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    Additive reductions in zebrafish PRPS1 activity result in a spectrum of deficiencies modeling several human PRPS1-associated diseases
    Springer Science and Business Media LLC ; 2016
    In:  Scientific Reports Vol. 6, No. 1 ( 2016-07-18)
    Details
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2016-07-18)
    Abstract: Phosphoribosyl pyrophosphate synthetase-1 ( PRPS1 ) is a key enzyme in nucleotide biosynthesis and mutations in PRPS1 are found in several human diseases including nonsyndromic sensorineural deafness, Charcot-Marie-Tooth disease-5 and Arts Syndrome. We utilized zebrafish as a model to confirm that mutations in PRPS1 result in phenotypic deficiencies in zebrafish similar to those in the associated human diseases. We found two paralogs in zebrafish, prps1a and prps1b and characterized each paralogous mutant individually as well as the double mutant fish. Zebrafish prps1a mutants and prps1a;prps1b double mutants showed similar morphological phenotypes with increasingly severe phenotypes as the number of mutant alleles increased. Phenotypes included smaller eyes and reduced hair cell numbers, consistent with the optic atrophy and hearing impairment observed in human patients. The double mutant also showed abnormal development of primary motor neurons, hair cell innervation and reduced leukocytes, consistent with the neuropathy and recurrent infection of the human patients possessing the most severe reductions of PRPS1 activity. Further analyses indicated the phenotypes were associated with a prolonged cell cycle likely resulting from reduced nucleotide synthesis and energy production in the mutant embryos. We further demonstrated the phenotypes were caused by delays in the tissues most highly expressing the prps1 genes.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    URL: Article
    DOI: 10.1038/srep29946
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
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  • 8
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    CRISPRz: a database of zebrafish validated sgRNAs
    Oxford University Press (OUP) ; 2016
    In:  Nucleic Acids Research Vol. 44, No. D1 ( 2016-01-04), p. D822-D826
    Details
    In: Nucleic Acids Research, Oxford University Press (OUP), Vol. 44, No. D1 ( 2016-01-04), p. D822-D826
    Type of Medium: Online Resource
    ISSN: 0305-1048 , 1362-4962
    URL: Article
    DOI: 10.1093/nar/gkv998
    RVK:
    WA 15000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2016
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  • 9
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    Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes
    American Society of Hematology ; 2020
    In:  Blood Vol. 136, No. Supplement 1 ( 2020-11-5), p. 32-32
    Details
    In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 32-32
    Abstract: ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2020-141043
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2020
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  • 10
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    A model of Costeff Syndrome reveals metabolic and protective functions of mitochondrial OPA3
    The Company of Biologists ; 2010
    In:  Development Vol. 137, No. 15 ( 2010-08-01), p. 2587-2596
    Details
    In: Development, The Company of Biologists, Vol. 137, No. 15 ( 2010-08-01), p. 2587-2596
    Abstract: Costeff Syndrome, which is caused by mutations in the OPTIC ATROPHY 3 (OPA3) gene, is an early-onset syndrome characterized by urinary excretion of 3-methylglutaconic acid (MGC), optic atrophy and movement disorders, including ataxia and extrapyramidal dysfunction. The OPA3 protein is enriched in the inner mitochondrial membrane and has mitochondrial targeting signals, but a requirement for mitochondrial localization has not been demonstrated. We find zebrafish opa3 mRNA to be expressed in the optic nerve and retinal layers, the counterparts of which in humans have high mitochondrial activity. Transcripts of zebrafish opa3 are also expressed in the embryonic brain, inner ear, heart, liver, intestine and swim bladder. We isolated a zebrafish opa3 null allele for which homozygous mutants display increased MGC levels, optic nerve deficits, ataxia and an extrapyramidal movement disorder. This correspondence of metabolic, ophthalmologic and movement abnormalities between humans and zebrafish demonstrates a phylogenetic conservation of OPA3 function. We also find that delivery of exogenous Opa3 can reduce increased MGC levels in opa3 mutants, and this reduction requires the mitochondrial localization signals of Opa3. By manipulating MGC precursor availability, we infer that elevated MGC in opa3 mutants derives from extra-mitochondrial HMG-CoA through a non-canonical pathway. The opa3 mutants have normal mitochondrial oxidative phosphorylation profiles, but are nonetheless sensitive to inhibitors of the electron transport chain, which supports clinical recommendations that individuals with Costeff Syndrome avoid mitochondria-damaging agents. In summary, this paper introduces a faithful Costeff Syndrome model and demonstrates a requirement for mitochondrial OPA3 to limit HMG-CoA-derived MGC and protect the electron transport chain against inhibitory compounds.
    Type of Medium: Online Resource
    ISSN: 1477-9129 , 0950-1991
    URL: Article
    DOI: 10.1242/dev.043745
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2010
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    detail.hit.zdb_id: 2007916-3
    SSG: 12
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