Belyantseva, I. A. | Search bioRxiv

bioRxiv2025

Methionine sulfoxide reductase B3 antioxidant activity is indispensable for mouse inner ear cuticular plate structure and hair bundle integrity

Methionine sulfoxide reductases (MSR) are enzymes responsible for catalyzing the reduction of methionine-sulfoxides. We previously demonstrated that variants in human MSRB3, a member of the MSR family, are associated with profound autosomal recessive prelingual non-syndromic hearing loss DFNB74. To better understand the role of MSRB3 in the auditory pathway, we generated a complete MsrB3 gene knock-out mouse model of human DFNB74 deafness. The MsrB3 deficient mouse showed profound hearing loss by postnatal day 16 (P16), which was accompanied by hair cell morphological abnormalities including altered stereocilia bundle shape and cuticular plate degeneration followed by hair cell apoptotic death. Although the absence of MSRB3 primarily affected the actin cytoskeleton, rootlets were present, and the expression and localization of major F-actin stereocilia-core proteins were unaltered. Biochemical assays demonstrated that wild-type MSRB3, but not MSRB3 harboring p.Cys89Gly, the same variant reported for human deafness DFNB74, can repolymerize oxidized actin. Consistent with these in vitro data, we observed a decreased ratio of reduced/total actin in the inner ears of Msrb3 knock-out mice. These data suggest a protective role of MSRB3 in the maintenance and maturation of stereocilia and hair cells, a conserved mechanism aimed at maintaining actin redox dynamics in these sensory cells.

genetics↗

bioRxiv2024

Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7As interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

cell biology↗

bioRxiv2017

A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency

Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations.\n\nAuthor SummaryPerrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.

genetics↗