Biology subjects

Vona, B.

Publications and source records attributed to Vona, B..

5 recordsLinked to original sources

bioRxiv2025

Protocol for a minigene splice assay using the pET01 vector

Aberrant splicing is recognized as a key contributor of hereditary disorders, yet characterizing the molecular effects of splice variants is an important task that poses several challenges. Here, we present a protocol for an in vitro splice assay using a minigene approach, which is especially useful when patient samples are not available for RNA analysis or when target genes or isoforms are not expressed in accessible tissues for direct analysis. We describe steps for assay design, including the cloning of minigene plasmids and subsequent transfection, followed by RNA isolation and cDNA synthesis. We also provide details on quantitative fragment analysis using capillary electrophoresis and optional subcloning to facilitate optimal sequencing of multiple splicing products.

genetics↗

bioRxiv2024

An inappropriate decline in ribosome levels drives a diverse set of neurodevelopmental disorders

Many neurodevelopmental defects are linked to perturbations in genes involved in housekeeping functions, such as those encoding ribosome biogenesis factors. However, how reductions in ribosome biogenesis can result in tissue and developmental specific defects remains a mystery. Here we describe new allelic variants in the ribosome biogenesis factor AIRIM primarily associated with neurodevelopmental disorders. Using human cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we identify a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, making differentiating cells particularly vulnerable to perturbations in ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing mTOR activity by both genetic and pharmacologic approaches ameliorates the growth and developmental defects associated with intellectual disability linked variants, identifying potential treatment options for specific brain ribosomopathies. This work reveals the cellular and molecular origins of protein synthesis defect-related disorders of human brain development. HighlightsO_LIAIRIM variants reduce ribosome levels specifically in neural progenitor cells. C_LIO_LIInappropriately low ribosome levels cause a transient delay in radial glia fate commitment. C_LIO_LIReduced ribosome levels impair translation of a selected subset of mRNAs. C_LIO_LIGenetic and pharmacologic activation of mTORC1 suppresses AIRIM-linked phenotypes. C_LI

developmental biology↗

bioRxiv2020

Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

genetics↗

bioRxiv2020

Radixin modulates stereocilia function and contributes to cochlear amplification

The stereocilia of the sensory cells in the inner ear contain high levels of the actin-binding protein radixin, encoded by the RDX gene. Radixin which is associated with mechanotransduction process such as PIP2 is known to be important for hearing but its functional role remains obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to directly visualize stereocilia motion while measuring the amplitude of the electrical potentials produced by sensory cells during acoustic stimulation. Experiments were performed in guinea pigs, where upon blocking radixin, a large decrease in sound-evoked electrical potentials occurred. Despite this decrease other important functional measures, such as electrically induced sensory cell motility and the sound-evoked deflections of stereocilia, showed a minor amplitude increase. This unique set of functional properties alterations demonstrate that radixin is necessary to ensure that the inner ear converts sound into electrical signals at acoustic rates. Radixin is therefore a necessary and important component of the cochlear amplifier, the energy-consuming process that boosts hearing sensitivity by up to 60 dB.

neuroscience↗

bioRxiv2019

Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion

The transport and Golgi organization 1 (TANGO1) family proteins have been shown to play pivotal roles in the secretory pathway. Full length TANGO1 is a transmembrane protein localised at endoplasmic reticulum exit sites (ERES), where it binds bulky cargo within the ER lumen and recruits membranes from the ER Golgi intermediate compartment (ERGIC) to create an exit route for their export. Tango1 knockout mice display a global collagen secretion defect and perinatal lethality. Here we report the first TANGO1-associated syndrome in humans, which mainly manifests in a collagenopathy. A synonymous substitution that results in exon 8 skipping in most mRNA molecules, ultimately leading to a truncated TANGO1 protein was identified as the disease-causing mutation. The four homozygously affected sons of a consanguineous family display severe dentinogenesis imperfecta, short stature, various skeletal abnormalities, insulin-dependent diabetes mellitus, sensorineural hearing loss, and mild intellectual disability. Functional studies in HeLa and U2OS cells revealed that the corresponding truncated TANGO1 protein is dispersed in the ER and its expression in cells with intact endogenous TANGO1 impairs cellular collagen I secretion.

genetics↗