RESUMO
ß-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2ß2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat ß-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in ß-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in ß-thalassemia.
Assuntos
Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Talassemia beta/genética , Proteínas Elk-1 do Domínio ets/genética , gama-Globinas/genética , Diferenciação Celular/genética , Linhagem Celular , Células Precursoras Eritroides/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Células K562 , Interferência de RNA , Talassemia beta/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , gama-Globinas/metabolismoRESUMO
The homozygous autosomal recessive truncating mutations of LDL receptor related protein associated protein 1 (LRPAP1) is a possible reason for Nonsyndromic Extreme Myopia, patients with which show typical chorioretinal degeneration. We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to study mechanisms of retinal degeneration underlying LRPAP1 deficiency with the help of the CRISPR/Cas9 system. Two distinct biallelic deletions in the cell line have been confirmed, which causing a frameshift and premature stop codons thus influence the translation of LRPAP1. FDCHDPe009-B has maintained normal stem cell morphology, pluripotent gene expression, parental karyotype, and ability to differentiate into three germ layers.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias Humanas , Sistemas CRISPR-Cas/genética , Linhagem Celular , Células-Tronco Embrionárias , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa DensidadeRESUMO
Methylation at the N6 position of adenosine (m(6)A) is the most abundant RNA modification within protein-coding and long noncoding RNAs in eukaryotes and is a reversible process with important biological functions. YT521-B homology domain family (YTHDF) proteins are the readers of m(6)A, the binding of which results in the alteration of the translation efficiency and stability of m(6)A-containing RNAs. However, the mechanism by which YTHDF proteins cause the degradation of m(6)A-containing RNAs is poorly understood. Here we report that m(6)A-containing RNAs exhibit accelerated deadenylation that is mediated by the CCR4-NOT deadenylase complex. We further show that YTHDF2 recruits the CCR4-NOT complex through a direct interaction between the YTHDF2 N-terminal region and the SH domain of the CNOT1 subunit, and that this recruitment is essential for the deadenylation of m(6)A-containing RNAs by CAF1 and CCR4. Therefore, we have uncovered the mechanism of YTHDF2-mediated degradation of m(6)A-containing RNAs in mammalian cells.
Assuntos
Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Adenosina/genética , Adenosina/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilação , Poliadenilação , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Receptores CCR4/genética , Receptores CCR4/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Fatores de Transcrição/genéticaRESUMO
Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16-18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3' end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3' overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications.