Identification of deubiquitinating enzyme genes relevant for the regulation of retina-specific genes

[eng] Protein post-translational modifications are regulatory mechanisms that cells use in response to intra- and extracellular signals. These signals modulate a panoply of conjugating enzymes that modify proteins post-translationally by the conjugation of a small functional group or peptide. The consequences of these modifications are very diverse, but they all present a common feature: they shift protein fate, localization or function. Besides, in the case of covalent protein modifications, such as ubiquitination, these regulatory mechanisms are reversible and dynamic. In mammals, only two E1 and around thirty E2 ligases have been described to participate in the ubiquitin cycle, in contrast to the approximately 600 E3 ligases identified. Note that this is a highly dynamic process and thus, cells also contain a group of deubiquitinating enzymes (DUBs), responsible for detaching ubiquitin from its substrates. DUBs process the ubiquitin precursor that has been transcribed from several genes as fusion proteins; deubiquitinate substrates and rescue them from protein degradation; and finally, they recycle Ub molecules from proteins committed to be degraded20. DUBs play important roles in disease and cellular processes; however and despite its evident importance in the organism, data on their mode of regulation and substrate specificity is still scarce21 The retina is the part of the eye responsible for capturing light stimuli from our surroundings. It is a neuronal tissue located at the posterior part of the eye, which captures light photons, converts the luminic energy into electrochemical stimuli and finally sends visual information to the brain where it is integrated. Vertebrate retina is formed by seven neuronal cell types organized in six precise functional and structural layers. The main function of the retina, the phototransduction, is carried out by two types of photoreceptor cells (PhR), cones and rods (see below). Photoreceptors are highly specialized cellular types, which differ both structurally and functionally, but that develop from the same photoreceptor progenitor precursor. Therefore, there must be a fine and tight genetic control to successfully achieve a properly structured and functional tissue. The regulation by photoreceptor-specific transcription factors has been amply described1,35,36; however, it is not their bare action that determines photoreceptor fate, since SUMO conjugation –and possibly other post-tranlational modifications, such ubiquitination– of TFs play a key role in this process37 In mammals, several comprehensive surveys of DUBs have been reported resulting in: in silico inventories of the DUBs in the human genome20,21; identification of protein interactors by cell-based proteomics analysis45; studies of subcellular localization46; functional involvement in maintaining genome integrity47. However, detailed expression and functional analysis for most DUBs on particular tissues or organs, such as the retina, is still missing. For these reasons, this work is intended as a study of deubiquitinating enzymes in the retina and their possible role in photoreceptor development and homeostasis. To that end, several objectives have been set and reached: 1. Analysis of the involvement of deubiquitinating enzymes (DUBs) in the mouse retina: I. Analysis of mRNA expression and pattern of expression via Real Time qPCR and in situ hybridization. II. Determination of the protein localization via fluorescent immunohistochemistry. III. Sequence and functional conservation analysis through phylogenetic and phenotypic studies. IV. Transcriptomic analysis of DUBs’ mRNA expression in the retina during developmentPreliminary analysis of the functional role of selected DUBs in mouse retinal development, via in vivo gene knockdown. 2. Devising an in-vivo cell system to study the role of DUB enzymes on the regulation of retinal promoters: I. Establishing a cell culture system of study II. Knockdown of DUB genes via shRNA and siRNA silencing techniques. 3. Identification of CRX post-translational modifications, particularly ubiquitynation.[spa] Las modificaciones post-traduccionales de proteínas son mecanismos de regulación que las células utilizan en respuestas a intra y extracelulares. En el caso de las modificaciones covalentes con proteínas, como lo es la ubiquitinación, estos mecanismos de regulación son reversibles y dinámicos. La ubiquitinación se hace reversible por la acción de las enzimas deubiquitinantes (DUBs), encargados de hidrolizar el enlace entre la ubiquitina y sus proteínas sustrato. Las DUBs juegan importantes roles en enfermedades y procesos celulares, no obstante, todavía hay poca información sobre su implicación y regulación de tejidos específicos, como la retina. La retina órgano de la visión responsable de capturar estímulos lumínicos, convertirlos en estímulos electroquímicos y finalmente mandar esta información hacia el córtex visual. Existen dos tipos de fotoreceptores, los conos y los bastones, que difieren funcional y estructuralmente, pero que provienen de una misma célula precursora. Se ha descrito que esta estricta diferenciación se lleva a cabo gracias a la acción combinada de factores de transcripción específicos junto a las modificaiones post-traduccionales, como lo és la molecula ubiquitin-like, SUMO. El presente trabajo pretende analizar la acción de los enzimas deubiquitinantes en el desarrollo de la retina y los fotoreceptores. Para ello, se ha llevado a cabo una primera descripción de los niveles y el patrón de expresión de estos enzimas en la retina; así como un anàlisis transcriptómico en diferentes estadíos de desarrollo retinal murino y humano. Además, se ha realizado un estudio de su conservación evolutiva y su implicación en fenotipos neuronales y de retina. Asimismo, se ha puesto a punto un sistema celular que replica condiciones fisiológicas similares a las retinales y se ha realizado un análisis de silenciamiento de los genes que codifican para las DUBs utilizando shRNA, siRNA y Gapmers. Finalmente, se ha realizado un análisis de las posibles modificaciones post-traduccionales en el factor de transcripción retinal CRX

Expression of deubiquitinating enzyme genes in the developing mammal retina

Purpose: Genes involved in the development and differentiation of the mammalian retina are also associated with inherited retinal dystrophies (IRDs) and age-related macular degeneration. Transcriptional regulation of retinal cell differentiation has been addressed by genetic and transcriptomic studies. Much less is known about the posttranslational regulation of key regulatory proteins, although mutations in some genes involved in ubiquitination and proteostasis E3 ligases and deubiquitinating enzymes (DUBs) cause IRDs. This study intends to provide new data on DUB gene expression during different developmental stages of mouse and human fetal retinas. Methods: We performed a comprehensive transcriptomic analysis of all the annotated human and mouse DUBs (87) in the developing mouse retina at several embryonic and postnatal time points compared with the transcriptome of the fetal human retina. An integrated comparison of data from transcriptomics, reported chromatin immunoprecipitation sequencing (ChIP-seq) of CRX and NRL transcription factors, and the phenotypic retinal alterations in different animal models is presented. Results: Several DUB genes are differentially expressed during the development of the mouse and human retinas in relation to proliferation or differentiation stages. Some DUB genes appear to be distinctly expressed during the differentiation stages of rod and cone photoreceptor cells, and their expression is altered in mouse knockout models of relevant photoreceptor transcription factors. We complemented this RNA-sequencing (RNA-seq) analysis with other reported expression and phenotypic data to underscore the involvement of DUBs in cell fate decision and photoreceptor differentiation. Conclusions: The present results highlight a short list of potential DUB candidates for retinal disorders, which require further study

Expression atlas of the Deubiquitinating enzymes in the adult mouse retina, their evolutionary diversification and phenotypic roles

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders