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1.
Nat Commun ; 15(1): 3138, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38605034

RESUMO

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.


Assuntos
Sítios de Splice de RNA , Retinose Pigmentar , Spliceossomos , Humanos , Spliceossomos/genética , Spliceossomos/metabolismo , Proteômica , Splicing de RNA/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Mensageiro/metabolismo , Mutação , DNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo
2.
J Invest Dermatol ; 142(2): 265-271.e1, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34762923

RESUMO

Volume scanning electron microscopy (VSEM) involves the serial sectioning and imaging of a sample using scanning electron microscopy (SEM), followed by segmentation and three-dimensional (3D) reconstruction using computer software packages to allow visualization of 3D structures. VSEM can reveal qualitative and quantitative properties of organelles and cells within tissues at nanoscale. The ability to visualize spatial relationships of structures of interest within and across cells in 3D space in particular sets VSEM apart from conventional SEM and transmission electron microscopy. Here, we provide an overview of VSEM platforms and image processing, highlighting characteristics that will aid selection of a method to address specific research questions in dermatological research.


Assuntos
Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Animais , Pesquisa Biomédica/instrumentação , Pesquisa Biomédica/métodos , Dermatologia/instrumentação , Dermatologia/métodos , Células HEK293 , Humanos , Imageamento Tridimensional/instrumentação , Microscopia Eletrônica de Varredura/instrumentação , Pele/citologia , Pele/diagnóstico por imagem , Pele/patologia
3.
Cell Rep ; 36(6): 109509, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34380033

RESUMO

The brain's ability to process complex information relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments-the soma, dendrites, and projecting axons-which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. In this study, we apply quantitative three-dimensional electron microscopy to map mitochondrial network morphology and complexity in the mouse brain. We examine somatic, dendritic, and axonal mitochondria in the dentate gyrus and cornu ammonis 1 (CA1) of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.


Assuntos
Envelhecimento/fisiologia , Axônios/fisiologia , Dendritos/fisiologia , Hipocampo/fisiologia , Mitocôndrias/metabolismo , Neurônios/citologia , Animais , Região CA1 Hipocampal/fisiologia , Giro Denteado/fisiologia , Feminino , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Frações Subcelulares/metabolismo
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