RESUMO
Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382 bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33 bp) of Frmpd1 binds to neural retina leucine zipper (NRL) and cone-rod homeobox protein (CRX), two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. Clustered regularly interspaced short palindromic repeats/Cas9-mediated deletion of the genomic region, including NRL and CRX binding sites, in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely expressed genes.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Domínios PDZ , Regiões Promotoras Genéticas , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Deleção de Sequência , Processamento Alternativo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/química , Diferenciação Celular , Éxons , Humanos , Ligação Proteica , Transcrição GênicaRESUMO
The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg(2+) and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg(2+) and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg(2+) may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg(2+) or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg(2+) toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg(2+)-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.
Assuntos
RNA Fúngico/química , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/química , Pareamento de Bases , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Compressor stations maintain pressure along natural gas pipelines to sustain gas flow. Unfortunately, they present human health concerns as they release chemical pollutants into the air, sometimes at levels higher than national air quality standards. Further, compressor stations are often placed in rural areas with higher levels of poverty and/or minority populations, contributing to environmental justice concerns. In this paper we investigate what chemical pollutants are emitted by compressor stations, the impacts of emitted pollutants on human health, and local community impacts. Based on the information gained from these examinations, we provide the following policy recommendations with the goal of minimizing harm to those affected by natural gas compressor stations: the Environmental Protection Agency (EPA) and relevant state agencies must increase air quality monitoring and data transparency; the EPA should direct more resources to monitoring programs specifically at compressor stations; the EPA should provide free indoor air quality monitoring to homes near compressor stations; the EPA needs to adjust its National Ambient Air Quality Standards to better protect communities and assess cumulative impacts; and decision-makers at all levels must pursue meaningful involvement from potentially affected communities. We find there is substantial evidence of negative impacts to strongly support these recommendations.