RESUMO
Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these splicing modulators.
Assuntos
Adenosina/química , Processamento Alternativo , Proteínas de Transporte/química , Fosfoproteínas/química , Fatores de Processamento de RNA/química , Proliferação de Células , Sobrevivência Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Compostos de Epóxi/química , Éxons , Álcoois Graxos/química , Células HCT116 , Humanos , Íntrons , Macrolídeos/química , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Piranos/química , Interferência de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Análise de Sequência de RNA , Compostos de Espiro/química , Spliceossomos/metabolismo , TransativadoresRESUMO
The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.
