Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability

Modeling splicing-related autosomal dominant retinitis pigmentosa in Caenorhabditis elegans

Mutations in some ubiquitously expressed splicing factors genes have been linked to the autosomal dominant form of a rare genetic disease called Retinitis Pigmentosa (adRP). RP is characterized by a progressive visual degeneration produced by the apoptosis of photoreceptors. Taking advantage of RNA mediated-interference (RNAi) and RNA-sequencing we started to build a C. elegans model where to study the disease. We found two important similarities between s-adRP and the RNAi phenotypes observed in C. elegans: (i) there is a cell-type-specific apoptosis and (ii) it seems to be associated with transcriptionally active tissues. We have stated a working model to investigate the mechanisms that triggers apoptosis in these s-adRP retinal cells. This model involves inefficient splicing, reduced transcriptional efficiency, and presence of R-loops as source of replicative stress and genomic instability. Additionally, we are using CRISPR/Cas9 to introduce specific s-adRP mutations in the C. elegans genome to constitute a platform where to screen for genetic or drug modifiers of the disease.Mutaciones en genes que codifican factores de splicing expresados de forma ubicua, han sido asociados a la forma autosómica dominante de una enfermedad genética rara llamada Retinitis Pigmentosa (adRP). La RP se caracteriza por una degeneración visual progresiva causada por la apoptosis de fotoreceptores. Utilizando técnicas de ARN de interferencia (RNAi) y secuenciación de ARN empezamos a construir un modelo de la enfermedad en C. elegans. Encontramos dos similitudes importantes entre la adRP asociada a splicing (s-adRP) y los fenotipos de RNAi en C. elegans: (i) hay una apoptosis específica de un tipo celular, y (ii) parece estar asociada a tejidos de alta actividad transcripcional. Establecimos un modelo para estudiar el mecanismo que desencadena la apoptosis en estas células de la retina afectaras por s-adRP. Este modelo incluye un splicing ineficiente, reducción en la eficiencia de la transcripción, y presencia de R-loops como fuente de estrés replicativo e inestabilidad genómica. Adicionalmente, estamos utilizando CRISPR/Cas9 para introducir mutaciones especificas de s-adRP en el genoma de C. elegans para constituir una plataforma donde identificar modificadores genéticos o farmacéuticos de la enfermedad

VEX1 Influences mVSG Expression During the Transition to Mammalian Infectivity in Trypanosoma brucei

International audienceThe Trypanosoma (T) brucei life cycle alternates between the tsetse fly vector and the mammalian host. In the insect, T. brucei undergoes several developmental stages until it reaches the salivary gland and differentiates into the metacyclic form, which is capable of infecting the next mammalian host. Mammalian infectivity is dependent on expression of the metacyclic variant surface glycoprotein genes as the cells develop into mature metacyclics. The VEX complex is essential for monoallelic variant surface glycoprotein expression in T. brucei bloodstream form, however, initiation of expression of the surface proteins genes during metacyclic differentiation is poorly understood. To better understand the transition to mature metacyclics and the control of metacyclic variant surface glycoprotein expression we examined the role of VEX1 in this process. We show that modulating VEX1 expression leads to a dysregulation of variant surface glycoprotein expression during metacyclogenesis, and that following both in vivo and in vitro metacyclic differentiation VEX1 relocalises from multiple nuclear foci in procyclic cells to one to two distinct nuclear foci in metacyclic cells - a pattern like the one seen in mammalian infective bloodstream forms. Our data suggest a role for VEX1 in the metacyclic differentiation process and their capacity to become infectious to the mammalian host

Mitophagy of polarized sperm-derived mitochondria after fertilization

International audienceLoss of membrane potential of sperm mitochondria has been regarded as the first step preceding mitophagy degradation after their entry into the C. elegans oocyte at fertilization. This is in line with the classical view of mitophagy of defective or abnormal mitochondria and could serve as a recognition signal for their specific and quick autophagy degradation. Here, using TMRE (tetramethylrhodamine ethyl ester) and live imaging we show that this is not the case. Instead, sperm inherited mitochondria show a stable labeling with TMRE before and at the time of autophagosomes formation. Interestingly, this labeling remains in late-stage-embryos of autophagy-defective-mutants suggesting that the loss of membrane potential occurs upon the entry of the mitochondria into the autophagy pathway. These stabilized and still polarized sperm mitochondria remain distinct but associated with the maternal-derived mitochondrial network suggesting a mechanism that prevents their fusion and represents an efficient additional protective system against fertilization-induced heteroplasmy

Fndc-1 contributes to paternal mitochondria elimination in C. elegans

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Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability