RESUMO
Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.
Assuntos
Espaço Intracelular/genética , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Células-Tronco/patologiaRESUMO
We identify a type of polycistronic transcript-derived long noncoding RNAs (lncRNAs) that are 5' small nucleolar RNA (snoRNA) capped and 3' polyadenylated (SPAs). SPA processing is associated with nascent mRNA 3' processing and kinetic competition between XRN2 trimming and Pol II elongation. Following cleavage/polyadenylation of its upstream gene, the downstream uncapped pre-SPA is trimmed by XRN2 until this exonuclease reaches the co-transcriptionally assembled snoRNP. This snoRNP complex prevents further degradation, generates a snoRNA 5' end, and allows continuous Pol II elongation. The imprinted 15q11-q13 encodes two SPAs that are deleted in Prader-Willi syndrome (PWS) patients. These lncRNAs form a nuclear accumulation that is enriched in RNA binding proteins (RBPs) including TDP43, RBFOX2, and hnRNP M. Generation of a human PWS cellular model by depleting these lncRNAs results in altered patterns of RBPs binding and alternative splicing. Together, these results expand the diversity of lncRNAs and provide additional insights into PWS pathogenesis.
Assuntos
Sequência de Bases , Síndrome de Prader-Willi/genética , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno/genética , Deleção de Sequência , Transcrição Gênica , Processamento Alternativo , Cromossomos Humanos Par 15 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Loci Gênicos , Impressão Genômica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Síndrome de Prader-Willi/metabolismo , Síndrome de Prader-Willi/patologia , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.