RESUMO
A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Proteínas/metabolismo , RNA/toxicidade , Adenosina Desaminase/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72 , Contagem de Células , Relação Dose-Resposta a Droga , Demência Frontotemporal/tratamento farmacológico , Demência Frontotemporal/genética , Ácido Glutâmico/toxicidade , Humanos , Células-Tronco Pluripotentes Induzidas , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Proteínas/genética , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA , Sequências Repetitivas de Ácido NucleicoRESUMO
BACKGROUND: Thousands of different long non-coding RNAs are expressed during embryonic development, but the function of these molecules remains largely unexplored. RESULTS: Here we characterize the expression and function of Six3OS, a long non-coding RNA that is transcribed from the distal promoter region of the gene encoding the homeodomain transcription factor Six3. Overexpression and knockdown analysis of Six3OS reveals that it plays an essential role in regulating retinal cell specification. We further observe that Six3OS regulates Six3 activity in developing retina, but does not do so by modulating Six3 expression. Finally, we show that Six3OS binds directly to Ezh2 and Eya family members, indicating that Six3OS can act as a molecular scaffold to recruit histone modification enzymes to Six3 target genes. CONCLUSIONS: Our findings demonstrate a novel mechanism by which promoter-associated long non-coding RNAs can modulate the activity of their associated protein coding genes, and highlight the importance of this diverse class of molecules in the control of neural development.
Assuntos
Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , RNA não Traduzido/genética , Retina/embriologia , Retina/metabolismo , Animais , Proteínas do Olho/metabolismo , Feminino , Células HEK293 , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Neurônios/citologia , Gravidez , Regiões Promotoras Genéticas/genética , RNA não Traduzido/metabolismo , Retina/citologia , Proteína Homeobox SIX3RESUMO
Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor estrogen-related receptor beta (ERRbeta) as selectively expressed in rod photoreceptors. Overexpression of ERRbeta induces expression of rod-specific genes in retinas of wild-type as well as Nrl(-/-) mice, which lack rod photoreceptors. Mutation of ERRbeta results in dysfunction and degeneration of rods, whereas inverse agonists of ERRbeta trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRbeta. ERRbeta coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRbeta activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRbeta is a critical regulator of rod photoreceptor function and survival, and suggest that ERRbeta agonists may be useful in the treatment of certain retinal dystrophies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Receptores de Estrogênio/metabolismo , Retina/embriologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transativadores/fisiologia , Animais , Sobrevivência Celular , Eletrorretinografia/métodos , Proteínas de Homeodomínio/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/metabolismo , Transativadores/metabolismoRESUMO
BACKGROUND: Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs) are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. RESULTS: We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. CONCLUSION: These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.
