Literatura académica sobre el tema "GPATCH11"

Disclosures Mutations in the core RNA splicing factor SF3B1 are common across MDS, CLL, and clonal hematopoiesis. Prior studies have elucidated that mutations in SF3B1 result in neomorphic widespread changes in splicing due to usage of aberrant intronic branchpoint nucleotides. However, the molecular basis by which mutant SF3B1 induces mis-splicing is not established and therapeutic means to correct mis-splicing due to mutant SF3B1 do not exist. Recently, the aberrant gain-of-function splicing activity of mutant SF3B1 was harnessed to regulate expression of proteins in a mutant selective manner using synthetic version of endogenous mRNA sequences uniquely recognized by mutant SF3B1. Here, we utilized this technology to engineer a synthetic intron derived from MAP3K7 that interrupts the coding sequence of the fluorescent protein mEmerald such that mutant SF3B1 produces lower mEmerald expression than SF3B1 wild-type (WT) cells (Fig.A). This fluorescent splicing reporter was then used to perform positive enrichment whole genome CRISPR screens to identify genes whose deletion corrects SF3B1 mutant aberrant splicing. GPATCH8 was the single most robust hit. GPATCH8 knockout strongly corrected mis-splicing of both synthetic and endogenous MAP3K7 introns (Fig.A). A previously unexplored protein, GPATCH8 has domains characteristic of RNA splicing factors including a G-patch motif, thought to be important in activating RNA helicases. Interestingly, recent work suggests that mutations in SF3B1 result in mis-splicing by disrupting physical interaction of SF3B1 to the RNA helicase DHX15 via the G-patch domain containing protein SUGP1. We therefore compared SUGP1 to GPATCH8 activity, evaluating first their transcriptome-wide binding sites using TRIBE-seq. We found that both proteins predominantly target intronic sequences over other genomic regions suggesting roles in splicing. RNA-seq of SF3B1 mutant and WT cells with or without GPATCH8/SUGP1 deletion revealed that ~33% of SF3B1 mutant splicing alterations are corrected by GPATCH8 deletion, while ~60% of SF3B1 mutant splicing alterations are recapitulated upon SUGP1 deletion. GPATCH8 regulated splicing events have stronger branchpoints indicating a potential role for GPATCH8 in 3' splice site recognition. To further understand the connection between GPATCH8 and mutant SF3B1, we performed mass spectrometry studies of immunoprecipitated endogenous GPATCH8 in SF3B1 WT and mutant knockin cells. This revealed strong interaction between GPATCH8 and DHX15. GPATCH8 interacts with DHX15 at the same sites previously shown to be occupied by SUGP1 in crystal structures of the SUGP1/DHX15 interaction. These data and further biochemical studies elucidated that GPATCH8 competes with SUGP1 for interaction to DHX15. As such, deletion of GPATCH8 corrects SF3B1 mutant mis-splicing by enhancing the interaction of SUGP1 and DHX15 to the mutant SF3b complex. Given that GPATCH8 is required for a large proportion of the splicing alterations induced by mutant SF3B1, we next evaluated the phenotypic effect of correcting SF3B1 mutant mis-splicing. We first tested the impact of anti-Gpatch8 shRNAs in bone marrow from WT mice as well as animals with conditional knockin of Sf3b1 K700E, K666N, or R625H mutations. While each Sf3b1 mutation results in impaired colony formation of hematopoietic precursors in methylcellulose assays, silencing of Gpatch8 rescued colony formation from hematopoietic precursors of each of the different Sf3b1 mutant mice and was tolerated by normal hematopoietic precursors (Fig.B). Moreover, while CRISPR base-edited knockin of the SF3B1 K700E mutation in adult CD34 + cells impaired erythroid development, this erythroid differentiation defect was rescued by GPATCH8 deletion in the same cells (Fig.B). These data identify GPATCH8 as a novel RNA splicing factor involved in quality control of RNA branchpoint selection whose expression is required for mis-splicing by the different mutant forms of SF3B1. GPATCH8 antagonizes the activity of SUGP1 by competing for interaction with DHX15. These findings suggest that disrupting GPATCH8/DHX15 interaction could have important therapeutic benefit for the multitude of SF3B1 mutant hematopoietic diseases. This study also demonstrates the power of synthetic intronic splicing assays for discovery of trans factors and druggable proteins required by leukemia-associated mutant RNA splicing factors. Abdel-Wahab: AbbVie, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Loxo/Lilly: Consultancy; Nurix Therapeutics: Research Funding; Minovia Therapeutics: Research Funding; Amphista Therapeutics: Consultancy; AstraZeneca: Consultancy; Harmonic Discovery: Current holder of stock options in a privately-held company. Figure 1

ObjectiveScleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc) strongly associated with anti-RNA polymerase III antibody (ARA) autoantibodies. We investigated genetic susceptibility and altered protein expression in renal biopsy specimens in ARA-positive patients with SRC.MethodsARA-positive patients (n = 99) with at least 5 years’ follow-up (49% with a history of SRC) were selected from a well characterized SSc cohort (n = 2254). Cases were genotyped using the Illumina Human Omni-express chip. Based on initial regression analysis, 9 single-nucleotide polymorphisms (SNP) were chosen for validation in a separate cohort of 256 ARA-positive patients (40 with SRC). Immunostaining of tissue sections from SRC or control kidney was used to quantify expression of candidate proteins based upon genetic analysis of the discovery cohort.ResultsAnalysis of 641,489 SNP suggested association of POU2F1 (rs2093658; P = 1.98 × 10−5), CTNND2 (rs1859082; P = 5.58 × 10−5), HECW2 (rs16849716; P = 1.2 × 10−4), and GPATCH2L (rs935332; P = 4.92 × 10−5) with SRC. Further, the validation cohort showed an association between rs935332 within the GPATCH2L region, with SRC (P = 0.025). Immunostaining of renal biopsy sections showed increased tubular expression of GPATCH2L (P = 0.026) and glomerular expression of CTNND2 (P = 0.026) in SRC samples (n = 8) compared with normal human kidney controls (n = 8), despite absence of any genetic replication for the associated SNP.ConclusionIncreased expression of 2 candidate proteins, GPATCH2L and CTNND2, in SRC compared with control kidney suggests a potential role in pathogenesis of SRC. For GPATCH2L, this may reflect genetic susceptibility in ARA-positive patients with SSc based upon 2 independent cohorts.

Abstract Genome-wide association studies have linked common variation in ZNF804A with an increased risk of schizophrenia. However, little is known about the biology of ZNF804A and its role in schizophrenia. Here, we investigate the function of ZNF804A using a variety of complementary molecular techniques. We show that ZNF804A is a nuclear protein that interacts with neuronal RNA splicing factors and RNA-binding proteins including RBFOX1, which is also associated with schizophrenia, CELF3/4, components of the ubiquitin-proteasome system and the ZNF804A paralog, GPATCH8. GPATCH8 also interacts with splicing factors and is localized to nuclear speckles indicative of a role in pre-messenger RNA (mRNA) processing. Sequence analysis showed that GPATCH8 contains ultraconserved, alternatively spliced poison exons that are also regulated by RBFOX proteins. ZNF804A knockdown in SH-SY5Y cells resulted in robust changes in gene expression and pre-mRNA splicing converging on pathways associated with nervous system development, synaptic contact, and cell adhesion. We observed enrichment (P = 1.66 × 10–9) for differentially spliced genes in ZNF804A-depleted cells among genes that contain RBFOX-dependent alternatively spliced exons. Differentially spliced genes in ZNF804A-depleted cells were also enriched for genes harboring de novo loss of function mutations in autism spectrum disorder (P = 6.25 × 10–7, enrichment 2.16) and common variant alleles associated with schizophrenia (P = .014), bipolar disorder and schizophrenia (P = .003), and autism spectrum disorder (P = .005). These data suggest that ZNF804A and its paralogs may interact with neuronal-splicing factors and RNA-binding proteins to regulate the expression of a subset of synaptic and neurodevelopmental genes.

In order to obtain unbiased results of target gene expression, selection of the most appropriate reference gene (RG) remains a key precondition. However, an experimental study focused on the validation of stably expressed RGs in the rat spinal cord (SC) during development or after spinal cord injury (SCI) is missing. In our study, we tested the stability of the expression of nine selected RGs in rat SC tissue during normal development (postnatal days 1–43, adulthood) and after minimal (mSCI) and contusion (cSCI) spinal cord injury. The following RGs were tested: common housekeeping genes of basal cell metabolism (Gapdh, Hprt1, Mapk6) and protein translation (Rpl29, Eef1a1, Eif2b2), as well as newly designed RGs (Gpatch1, Gorasp1, Cds2) selected according to the RefGenes tool of GeneVestigator. The stability of RGs was assessed by geNorm, NormFinder, and BestKeeper. All three applets favored Gapdh and Eef1a1 as the most stable genes in SC during development. In both models of SCI, Eif2b2 displayed the highest stability of expression, followed by Gapdh and Gorasp1/Hprt1 in cSCI, and Gapdh and Eef1a1 in the mSCI experiments. To verify our results, selected RGs were employed for normalization of the expression of genes with a clear biological context in the SC—Gfap and Slc1a3/Glast during postnatal development and Aif1/Iba1 and Cd68/Ed1 after SCI.

Background/Objectives: We investigated maternal and parent-of-origin (PoO) gene-environment interaction effects on the risk of nonsyndromic orofacial clefts for two maternal environmental factors: periconceptional smoking and folic acid supplementation. Methods: Genome-wide single nucleotide polymorphisms (SNPs) genotypes and TopMed-imputed genotypes were obtained for case-parent triads from the EUROCRAN and ITALCLEFT studies. Candidate regions were selected around target SNPs from a previous genome-wide association study, resulting in 12 (726 SNPs) and 11 regions (730 SNPs) for maternal and PoO effects, respectively. Log-linear models were used to analyze 404 case-parent triads and 40 case-parent dyads. p-values were combined across regions. Results: None of the interactions reached statistical significance after correction for the number of regions tested. Nominally significant (pooled p-values < 0.05) interactions pointed to regions in or close to genes LRRC7 (maternal gene-folate interaction), NCKAP5 (PoO-smoking interaction), and IFT43 and GPATCH2L (PoO-folate interaction). Conclusions: Our results suggested that the genetic effects in or around these genes were heightened under periconceptional exposure to tobacco or no folic acid supplementation. The involvement of these genes in orofacial cleft development, in conjunction with environmental exposures, should be further studied.

Abstract The transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is thought to be associated with changes in immune processes. We have therefore used serologic analysis of recombinant cDNA expression library to screen the sera of MGUS patients to identify tumor-associated antigens. A total of 10 antigens were identified, with specific antibody responses in MGUS. Responses appeared to be directed against intracellular proteins involved in cellular functions, such as apoptosis (SON, IFT57/HIPPI), DNA and RNA binding (ZNF292, GPATCH4), signal transduction regulators (AKAP11), transcriptional corepressor (IRF2BP2), developmental proteins (OFD1), and proteins of the ubiquitin-proteasome pathway (PSMC1). Importantly, the gene responsible for the oral-facial-digital type I syndrome (OFD1) had response in 6 of 29 (20.6%) MGUS patients but 0 of 11 newly diagnosed MM patients. Interestingly, 3 of 11 (27.2%) MM patients after autologous stem cell transplantations showed responses to OFD1. We have confirmed T-cell responses against OFD1 in MGUS and observed down-regulation of GLI1/PTCH1 and p-β-catenin after OFD1 knock-down with specific siRNA, suggesting its functional role in the regulation of Hh and Wnt pathways. These findings demonstrate OFD1 as an important immune target and highlight its possible role in signal transduction and tumorigenesis in MGUS and MM.

<p><em>Spodoptera exigua</em> (Hübner) are nocturnal insects and one of the important pests of shallot. The activities of nocturnal insects are influenced by the moonlight. The aim of this research is to analyze the effect of moonlight on oviposition behaviour: number of egg patches, number of eggs per patches and egg gpatches distribution pattern of <em>S. exigua</em>. The research was done on three shallot field at Brebes Regency, Central Java during the new, half and full moon. The sampling included observing the number of egg groups, the number of eggs per egg group, and egg groups distribution pattern of <em>S. exigua</em> on each observation plot. The egg groups distribution pattern of <em>S. exigua</em> based on Morisita index. The result of this research showed that the number of egg groups and the number of eggs per egg group always increased from the new, half, and full moon. In general, the egg groups distribution pattern of <em>S. exigua</em> were clustered on three moonlight conditions. High intensity of moonlight (full moon) was be able to increase the number of egg groups and the number of eggs per egg groups of <em>S. exigua</em>, its potentially cause more attacks.</p>

Les dystrophies rétiniennes sévères et précoces (EOSRD) et l'amaurose congénitale de Leber (ACL - MIM204000) sont les principales causes de cécité incurable chez les enfants. Ces maladies, variables sur le plan clinique, génétique et physiopathologique, peuvent être le signe de syndromes multisystémiques, tels que les ciliopathies. Elles se transmettent le plus souvent de manière autosomique récessive et l'implication de plusieurs gènes a été confirmée. Cependant, l'histoire et l'expression clinique de l'ACL sont imparfaitement comprises et de nombreuses mutations restent inconnues. Il est nécessaire de continuer à déchiffrer ces aspects pour affiner la compréhension de la physiopathologie. L'identification de nouveaux gènes responsables et les corrélations génotype-phénotype sont essentiels à la prise en charge des patients. Grâce au séquençage à haut débit des gènes connus de l'ACL/EOSRD et aux investigations dans les centres de référence cliniques, le Laboratoire de génétique ophtalmologique (LGO) a identifié les causes moléculaires de la maladie dans plus de 80 % des cas dans une cohorte de plus de 700 familles. À ce jour, 40 familles de ACL/EOSRD non résolues ont été soumises au séquençage de l'exome entier (WES), ce qui a permis d'identifier des gènes candidats, sélectionnés en vue d'une validation fonctionnelle. Des variants délétères de GPATCH11 ont été identifiés dans six familles comprenant 12 individus atteints de dystrophie rétinienne, présentant des troubles neurologiques et des anomalies squelettiques, fournissant des arguments forts que des mutations récessives dans le gène GPATCH11 sont responsables de la maladie. GPATCH11 est l'une des protéines contenant le domaine G-patch la moins étudiée, connues pour contribuer au spliceosome. Quatre mutations récessives ont été identifiées, avec la mutation du site d'épissage NM_174931.4 : c.328+1G>T commune à quatre familles sur six et affectant le site d'épissage consensus de l'intron 4, ce qui entraîne l'exclusion de l'exon 4 du transcrit sans rupture du cadre de lecture, produisant ainsi une protéine plus courte. La protéine GPATCH11, dans sa forme sauvage ou mutée, est localisée à la fois, de façon diffuse dans le nucléoplasme et dans le centrosome des cils primaires des fibroblastes, suggérant des rôles dans le métabolisme de l'ARN et des cils. Le modèle de souris (Gpatch11delta5/delta5) généré à l'Institut Imagine, portant la délétion de l'exon 5 équivalent à l'exon 4 de GPATCH11 humain, reproduit les défauts phénotypiques des patients, avec la présence d'une dystrophie rétinienne et des anomalies comportementales. Le transcriptome de la rétine a identifié des voies dérégulées dans l'expression et l'épissage des gènes, impactant des processus clés tels que les réponses à la lumière des photorécepteurs, la régulation de l'ARN et le métabolisme associé aux cils primaires. L'analyse par spectrométrie de masse a trouvé des protéines régulées à la baisse impliquées dans la perception visuelle, la fonction synaptique et les mécanismes de liaison et d'épissage de l'ARN, et des protéines régulées à la hausse impliquées principalement dans le métabolisme et l'épissage de l'ARN (Publication 1). En outre, l'implication de GPATCH11 dans le cerveau est en cours d'exploration par immunomarquage et analyse transcriptomique/protéomique, en se concentrant sur l'hippocampe, structure cérébrale responsable de la mémoire. Les souris Gpatch11delta5/delta5 sont viables et se développent normalement, toutefois les mâles sont complètement infertiles et présentent des testicules plus petits que la normale et vides, dont la cause est en cours d'étude en collaboration avec un laboratoire externe (Part 2A, B)
Early onset retinal dystrophies (EOSRD) and Leber congenital amaurosis (LCA - MIM204000) are the leading cause of incurable blindness in children. These diseases, clinically, genetically, and pathophysiologically variable, can be the sign of multisystemic syndromes, such as ciliopathies. They are mostly inherited in autosomal recessive manner, and several genes have been confirmed to be involved. However, the history and clinical expression of LCA are imperfectly understood and many mutations remain unknown. There is a need to continue deciphering these aspects to refine the understanding of pathophysiology. The identification of new responsible genes and the genotype-phenotype correlations are essential for disease management. Thanks to high-throughput gene panel-based sequencing of known LCA/EOSRD genes and investigation in clinical reference centres, the Laboratory of Genetics in Ophthalmology (LGO) has identified the molecular causes of the disease in more than 80% of cases in a cohort of over 700 families. To date, 40 unresolved LCA/EOSRD families have been submitted to whole exome sequencing (WES), leading to the identification of candidate genes, which have been selected for functional validation. Deleterious GPATCH11 variants have been identified in six families comprising 12 affected individuals with retinal dystrophy, exhibiting neurological disorders and skeletal anomalies, providing compelling evidence that recessive mutations in the GPATCH11 gene are responsible for the disease. GPATCH11 is one of the lesser-explored G-patch domain containing proteins, which are known to contribute to the spliceosome. Four recessive mutations were identified, with the splice-site NM_174931.4: c.328+1G>T being common to four out of six families and affecting the consensus splice site of intron 4, causing exon 4 to be excluded from the transcript without breaking the reading frame and producing a shorter protein. Both wild-type and mutated GPATCH11 proteins are localised in the nucleoplasm with a diffuse pattern and in the centrosome of the primary cilia of fibroblasts, suggesting roles in RNA and cilia metabolism. The mouse model (Gpatch11delta5/delta5) generated at the Institute Imagine, carrying the deletion of exon 5 equivalent to exon 4 of human GPATCH11, replicates the patients' phenotypic defects, such as retinal dystrophy and behavioural abnormalities. Retina transcriptome analysis identified deregulated pathways in gene expression and splicing, impacting key processes, such as photoreceptor light responses, RNA regulation, and primary cilia-associated metabolism. Mass-spectrometry analysis found downregulated proteins involved in vision perception, synaptic function and RNA binding and splicing pathways, and upregulated proteins mostly involved in RNA processing and splicing (Publication 1). Furthermore, the involvement of GPATCH11 in the brain is currently being explored through immunostaining and transcriptome/proteome analysis, focusing on the hippocampus, a brain structure responsible for memory. Gpatch11delta5/delta5 mice are viable and develop normally, except that males are completely infertile and exhibit smaller than normal and empty testis. The cause of this infertility is under investigation in collaboration with an external laboratory (Part 2A, B)