RESUMO
Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical 'terminator' gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a 'moonlighting' gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2's novel dual role tunes mitochondrial gene expression in either direction during development.
Assuntos
Proteínas de Protozoários , Edição de RNA , RNA Mensageiro , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Mitocôndrias/genética , RNA Guia de Sistemas CRISPR-Cas/genética , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismoRESUMO
U-insertion/deletion (U-indel) RNA editing in trypanosome mitochondria is directed by guide RNAs (gRNAs). This editing may developmentally control respiration in bloodstream forms (BSF) and insect procyclic forms (PCF). Holo-editosomes include the accessory RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C), but the specific proteins controlling differential editing remain unknown. Also, RNA editing appears highly error prone because most U-indels do not match the canonical pattern. However, despite extensive non-canonical editing of unknown functions, accurate canonical editing is required for normal cell growth. In PCF, REH2C controls editing fidelity in RESC-bound mRNAs. Here, we report that KREH2, a REH2C-associated helicase, developmentally controls programmed non-canonical editing, including an abundant 3' element in ATPase subunit 6 (A6) mRNA. The 3' element sequence is directed by a proposed novel regulatory gRNA. In PCF, KREH2 RNAi-knockdown up-regulates the 3' element, which establishes a stable structure hindering element removal by canonical initiator-gRNA-directed editing. In BSF, KREH2-knockdown does not up-regulate the 3' element but reduces its high abundance. Thus, KREH2 differentially controls extensive non-canonical editing and associated RNA structure via a novel regulatory gRNA, potentially hijacking factors as a 'molecular sponge'. Furthermore, this gRNA is bifunctional, serving in canonical CR4 mRNA editing whilst installing a structural element in A6 mRNA.
Assuntos
Trypanosoma brucei brucei , Trypanosoma , RNA Mensageiro/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma/genética , RNA/genética , RNA de Protozoário/genética , RNA de Protozoário/metabolismoRESUMO
Health plans play an important role in tobacco control. In this chapter we present an overview of the scientific research on health plan involvement in clinical and community interventions regarding tobacco use. Also included are interventions that have been undertaken by health plans to lower smoking rates among their members and the general population. We conclude with a new model that can be used to engage health plans in tobacco control efforts and a case study that outlines how one health plan has implemented this new model.