A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5′ untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes

RIBOSOMAL LESIONS PROMOTE ONCOGENIC MUTAGENESIS

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Ribosomal Lesions Promote Oncogenic Mutagenesis

Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S-associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation. SIGNIFICANCE: Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.status: publishe

Generation and Characterization of an Nxf7 Knockout Mouse to Study NXF5 Deficiency in a Patient with Intellectual Disability

Members of the Nuclear eXport Factor (NXF) family are involved in the export of mRNA from the nucleus to the cytoplasm, or hypothesized to play a role in transport of cytoplasmic mRNA. We previously reported on the loss of NXF5 in a male patient with a syndromic form of intellectual disability. To study the functional role of NXF5 we identified the mouse counterpart. Based on synteny, mouse Nxf2 is the ortholog of human NXF5. However, we provide several lines of evidence that mouse Nxf7 is the actual functional equivalent of NXF5. Both Nxf7 and NXF5 are predominantly expressed in the brain, show cytoplasmic localization, and present as granules in neuronal dendrites suggesting a role in cytoplasmic mRNA metabolism in neurons. Nxf7 was primarily detected in the pyramidal cells of the hippocampus and in layer V of the cortex. Similar to human NXF2, mouse Nxf2 is highly expressed in testis and shows a nuclear localization. Interestingly, these findings point to a different evolutionary path for both NXF genes in human and mouse. We thus generated and validated Nxf7 knockout mice, which were fertile and did not present any gross anatomical or morphological abnormalities. Expression profiling in the hippocampus and the cortex did not reveal significant changes between wild-type and Nxf7 knockout mice. However, impaired spatial memory was observed in these KO mice when evaluated in the Morris water maze test. In conclusion, our findings provide strong evidence that mouse Nxf7 is the functional counterpart of human NXF5, which might play a critical role in mRNA metabolism in the brain.status: publishe

Development of a controller platform for educational projects: A case study

Due to the huge growth of the electronics industry, an increasing shortage of electronic engineers is experienced. The authors believe that educational projects, connecting electronics to real-life problems and applications, can convince students to choose for an education in the field of electronics. Furthermore, most students have insufficient insight in the contents of their studies at the moment they have to make their choice. A positive experience, for instance in the form of a team game, can have a huge impact on students’ career choices [1]. The game contains several technical challenges and allows young people to experience and understand the possibilities and huge variety within the field of electronics.status: publishe

Early Frameshift Mutation in PIGA Identified in a Large XLID Family Without Neonatal Lethality

The phosphatidylinositol glycan class A (PIGA) protein is a member of the glycosylphosphatidylinositol (GPI) anchor pathway. Germline mutations in PIGA located at Xp22.2 are thought to be lethal in males. However, a nonsense mutation in the last coding exon was recently described in two brothers with multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) who survived through birth likely due to the hypomorphic nature of the truncated protein, but died in their first weeks of life. Here, we report on a frameshift mutation early in the PIGA cDNA (c.76dupT; p.Y26Lfs*3) that co-segregates with the disease in a large family diagnosed with a severe syndromic form of X-linked intellectual disability (XLID). Unexpectedly, CD59 surface expression suggested the production of a shorter PIGA protein with residual functionality. We provide evidence that the second methionine at position 37 may be used for the translation of a 36 amino acids shorter PIGA. Complementation assays confirmed that this shorter PIGA cDNA was able to partially rescue the surface expression of CD59 in a PIGA-null cell line. Taken together, our data strongly suggest that the early frameshift mutation in PIGA produces a truncated hypomorph, which is sufficient to rescue the lethality in males but not the MCAHS2-like phenotype. This article is protected by copyright. All rights reserved.status: publishe

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells

none17siMammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.openVerbelen B.; Girardi T.; Sulima S.O.; Vereecke S.; Verstraete P.; Verbeeck J.; Royaert J.; Cinque S.; Montanaro L.; Penzo M.; Imbrechts M.; Geukens N.; Geuens T.; Dierckx K.; Pepe D.; Kampen K.; De Keersmaecker K.Verbelen B.; Girardi T.; Sulima S.O.; Vereecke S.; Verstraete P.; Verbeeck J.; Royaert J.; Cinque S.; Montanaro L.; Penzo M.; Imbrechts M.; Geukens N.; Geuens T.; Dierckx K.; Pepe D.; Kampen K.; De Keersmaecker K