title: PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly.
abstract: 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 2α (eIF2α). We demonstrate a novel, positive role for PKR activation and eIF2α phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on β-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2α on Serine 51. Nonphosphorylatable mutant eIF2αS51A blocked β-globin mRNA splicing in cells and nuclear extract. Mutations of the β-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 β-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 β-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. α-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 eIF2α phosphorylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Lena
authors.initials: L
authors.lastname: Ilan
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Farhat
authors.initials: F
authors.lastname: Osman
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Lise Sarah
authors.initials: LS
authors.lastname: Namer
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Einav
authors.initials: E
authors.lastname: Eliahu
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Smadar
authors.initials: S
authors.lastname: Cohen-Chalamish
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Ben-Asouli
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Yona
authors.initials: Y
authors.lastname: Banai
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Raymond
authors.initials: R
authors.lastname: Kaempfer
mesh_terms: D001483:Base Sequence; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D008954:Models, Biological; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D017381:Spliceosomes / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005914:Globins; D019892:eIF-2 Kinase