PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly

Short elements in mammalian mRNA can control gene expression by activating the RNA-dependent protein kinase PKR that attenuates translation by phosphorylating cytoplasmic eukaryotic initiation factor 2 alpha (eIF2 alpha). We demonstrate a novel, positive role for PKR activation and eIF2 alpha phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on beta-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2 alpha on Serine 51. Nonphosphorylatable mutant eIF alpha S51A blocked beta-globin mRNA splicing in cells and nuclear extract. Mutations of the beta-globin RNA activator abrogated PKR activation and profoundly affected mRNA splicing efficiency. PKR depletion abrogated splicing and spliceosome assembly; recombinant PKR effectively restored splicing. Excision of the first intron of beta-globin induces strand displacement within the RNA activator of PKR by a sequence from exon 2, a structural rearrangement that silences the ability of spliced beta-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. a-Globin pre-mRNA splicing is controlled likewise but positions of PKR activator and silencer are reversed, demonstrating evolutionary flexibility in how PKR activation regulates globin mRNA splicing through eIF2 alpha phosphorylation.