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PKR activation and eIF2? 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 2? (eIF2?). We demonstrate a novel, positive role for PKR activation and eIF2? phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on ?-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2? on Serine 51. Nonphosphorylatable mutant eIF2?S51A blocked ?-globin mRNA splicing in cells and nuclear extract. Mutations of the ?-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 ?-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 ?-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. ?-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? phosphorylation.

SUBMITTER: Ilan L

PROVIDER: S-EPMC5520854 | biostudies-other | 2017 May

REPOSITORIES: biostudies-other

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

Ilan Lena L Osman Farhat F Namer Lise Sarah LS Eliahu Einav E Cohen-Chalamish Smadar S Ben-Asouli Yitzhak Y Banai Yona Y Kaempfer Raymond R

Cell research 20170404 5

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 ...[more]

PMID: 28374749

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Project description:Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress. A microarray analysis from our laboratory identified gene transcripts suggested to be under translation control in mouse embryonic fibroblast (MEF) cells following a 6 hour treatment with thapsigargin, a potent inducer of ER stress, or no stress. The mRNAs were separated by sucrose gradient analyses to yield three fractions, those transcripts associated with large polysomes (?4 ribosomes per mRNA), those associated with monosome, disomes, or trisomes, and those fractionated at the top of the gradient with free ribosomes. RNA was extracted from sucrose gradients corresponding to these fractions and hybridized on Affymetrix microarrays. In parallel, we also measured total levels for each gene transcript in the presence or absence of thapsigargin treatment to address transcription regulation coincident with translational control. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples.

Dataset Information

PKR activation and eIF2? phosphorylation mediate human globin mRNA splicing at spliceosome assembly.

Publications

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

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