Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD

A comprehensive functional study of Caenorhabditis elegans rsr-2 uncovers a new link between splicing and transcription

Durant el transcurs d’un rastreig a gran escala per RNA d’interferència (iARN) en Caenorhabditis elegans, el gen rsr-2 va ser identificat com a interactor genètic del gen lin-35 Retinoblastoma, l’homòleg de la família humana de Retinoblastoma. El gen rsr-2 és l’ortòleg de la proteina humana d’splicing SRm300/SRRM2. A diferència del seu ortòleg en llevat Cwc21, rsr-2 és essencial per la viabilitat. Degut a que la forta inactivació d’rsr-2 produeix uns fenotips molt severs, hem aprofitat l’efecte lleu que produeix l’ARN interferent a través de l’aliment per estudiar les funcions d’rsr-2 durant el desenvolupament. Els assatjos d’iARN d’rsr-2 juntament amb anàlisis d’epistàsia genètica situen rsr-2 en la via de la determinació sexual de la línia germinal. Tot i així, tiling arrays d’animals rsr-2(iARN) no han revelat defectes significatius en l’splicing. Gràcies a experiments d’immunofluorescència, hem observat que un anticòs específic per RSR-2 co-localitza amb cromatina en nuclis de cèl·lules de la línia germinal. Interessantment, experiments d’immunoprecipitació de cromatina i seqüenciació massiva (ChIP-Seq) han desvetllat que RSR-2 co-precipita cromatina seguint un patró similar al de l’ARN polimerassa II. Aquests experiments de ChIP-Seq també han fet palès que RSR-2 és reclutada a la cromatina d’un mode que és independent de l’splicing i suggereixen que RSR-2 podria desenvolupar un rol de regulador transcripcional. Addicionalment, hem explorat els transcriptomes d’animals rsr-2(iARN) i prp-8(iARN) en estadi de larva 3 (L3), fet que ens ha permès classificar rsr-2 com a un factor d’splicing no essencial. Conjuntament, el nostre estudi mostra que RSR-2 és una proteina multifuncional que regula el desenvolupament de Caenorhabditis elegans influenciant, i probablement acoblant, els processos d’splicing i transcripció.During the course of a large scale interference RNA (RNAi) screen in Caenorhabditis elegans, rsr-2 was identified as a genetic interactor of lin-35 Rb, the homolog of human Retinoblastoma. The rsr-2 gene encodes the ortholog of the human spliceosomal protein SRm300/SRRM2. In contrast to its yeast ortholog Cwc21, rsr-2 is essential for viability. Since strong inactivation of rsr-2 produces severe phenotypes, we took advantage of the mild effect of RNAi by feeding to study functions of rsr-2 during development. rsr-2 RNAi assays and genetic epistasis analysis locate rsr-2 within the germ line sex determination pathway but tiling arrays of rsr-2(RNAi) animals do not disclose significant splicing defects. By inmunofluorescence, we observe that an antibody specific for RSR-2 co-localizes with chromatin in germ line nuclei. Interestingly, ChIP-Seq experiments reveal that RSR-2 co-precipitates chromatin in a pattern similar to that of RNA Polymerase II. These ChIP-Seq assays also evidenced a splicing-independent recruitment of RSR-2 to chromatin and suggest that RSR-2 could have a role in regulating transcription. Moreover, we have explored the transcriptomes of rsr-2(RNAi) and prp-8(RNAi) L3 worms by RNA-Seq, which classified rsr-2 as a non-essential splicing factor. Altogether, our study shows that RSR-2 is a multifunctional protein that regulates development by influencing, and probably coupling, splicing and transcription

A comprehensive functional study of Caenorhabditis elegans rsr-2 uncovers a new link between splicing and transcription

Durant el transcurs d'un rastreig a gran escala per RNA d'interferència (iARN) en Caenorhabditis elegans, el gen rsr-2 va ser identificat com a interactor genètic del gen lin-35 Retinoblastoma, l'homòleg de la família humana de Retinoblastoma. El gen rsr-2 és l'ortòleg de la proteina humana d'splicing SRm300/SRRM2. A diferència del seu ortòleg en llevat Cwc21, rsr-2 és essencial per la viabilitat. Degut a que la forta inactivació d'rsr-2 produeix uns fenotips molt severs, hem aprofitat l'efecte lleu que produeix l'ARN interferent a través de l'aliment per estudiar les funcions d'rsr-2 durant el desenvolupament. Els assatjos d'iARN d'rsr-2 juntament amb anàlisis d'epistàsia genètica situen rsr-2 en la via de la determinació sexual de la línia germinal. Tot i així, tiling arrays d'animals rsr-2(iARN) no han revelat defectes significatius en l'splicing. Gràcies a experiments d'immunofluorescència, hem observat que un anticòs específic per RSR-2 co-localitza amb cromatina en nuclis de cèl·lules de la línia germinal. Interessantment, experiments d'immunoprecipitació de cromatina i seqüenciació massiva (ChIP-Seq) han desvetllat que RSR-2 co-precipita cromatina seguint un patró similar al de l'ARN polimerassa II. Aquests experiments de ChIP-Seq també han fet palès que RSR-2 és reclutada a la cromatina d'un mode que és independent de l'splicing i suggereixen que RSR-2 podria desenvolupar un rol de regulador transcripcional. Addicionalment, hem explorat els transcriptomes d'animals rsr-2(iARN) i prp-8(iARN) en estadi de larva 3 (L3), fet que ens ha permès classificar rsr-2 com a un factor d'splicing no essencial. Conjuntament, el nostre estudi mostra que RSR-2 és una proteina multifuncional que regula el desenvolupament de Caenorhabditis elegans influenciant, i probablement acoblant, els processos d'splicing i transcripció.During the course of a large scale interference RNA (RNAi) screen in Caenorhabditis elegans, rsr-2 was identified as a genetic interactor of lin-35 Rb, the homolog of human Retinoblastoma. The rsr-2 gene encodes the ortholog of the human spliceosomal protein SRm300/SRRM2. In contrast to its yeast ortholog Cwc21, rsr-2 is essential for viability. Since strong inactivation of rsr-2 produces severe phenotypes, we took advantage of the mild effect of RNAi by feeding to study functions of rsr-2 during development. rsr-2 RNAi assays and genetic epistasis analysis locate rsr-2 within the germ line sex determination pathway but tiling arrays of rsr-2(RNAi) animals do not disclose significant splicing defects. By inmunofluorescence, we observe that an antibody specific for RSR-2 co-localizes with chromatin in germ line nuclei. Interestingly, ChIP-Seq experiments reveal that RSR-2 co-precipitates chromatin in a pattern similar to that of RNA Polymerase II. These ChIP-Seq assays also evidenced a splicing-independent recruitment of RSR-2 to chromatin and suggest that RSR-2 could have a role in regulating transcription. Moreover, we have explored the transcriptomes of rsr-2(RNAi) and prp-8(RNAi) L3 worms by RNA-Seq, which classified rsr-2 as a non-essential splicing factor. Altogether, our study shows that RSR-2 is a multifunctional protein that regulates development by influencing, and probably coupling, splicing and transcription

Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance