1. Volume 20, Issue 1, Pages 188-200 (July 2017)
An Ancient Pseudoknot in TNF-α Pre-mRNA Activates PKR, Inducing eIF2α Phosphorylation that Potently Enhances Splicing 
Lise Sarah Namer, Farhat Osman, Yona Banai, Benoît Masquida, Rodrigo Jung, Raymond Kaempfer 
Cell Reports 
Volume 20, Issue 1, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 188-200DOI: (10.1016/j.celrep.2017.06.035)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Structural and Mutational Analysis of the TNF RNA Element that Renders Splicing Dependent on PKR
(A and B) Knockdown of PKR inhibits TNF mRNA splicing. HeLa cells stably expressing non-complementary (NC) or PKR-complementary shRNA (KD) were transfected with TNF-β gene expression vector lacking a 2-APRE (TNF-β) or harboring the TNF-α 2-APRE in the 3′ UTR (TNF-β/2-APRE). Spliced and unspliced TNF-β transcripts were determined at intervals by real-time qPCR; splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3) (A). Western blots show total PKR in wild-type (WT), NC, and KD cells, with actin as the loading control (left) and total and phosphorylated eIF2α (P-eIF2α) (right) (B).
(C) Secondary structure of human 2-APRE RNA, supported by footprinting, nuclease sensitivity mapping, and mutagenesis. Colors show protection from nuclease attack by rPKR. Pseudoknot stems P1 (boxed) and P2, helices S1–S3, linker (lin) and connector (con) between S2 and the remainder of the 2-APRE are indicated.
(D–F) A U-A pair at the base of 2-APRE helix S2 is critical for PKR activation, splicing efficiency, and protein yield. Mutated bases are in red. Activation of rPKR by WT or mutant 2-APRE RNA is shown. LI, labeling intensity of PKR band in autoradiogram (percentage of WT for each RNA concentration, 0.05 and 0.1 ng/μL) (D and F). For BHK-21 cells transfected with TNF-β gene vector harboring WT or mutant 2-APRE in the 3′ UTR, splicing efficiency was determined at 30 hr post-transfection and secreted TNF-β at 48 hr; TNF-β/mRNA ratio denotes translation efficiency (error bars, SEM; n = 3) and PKR activation (LI) is from (D) for 0.05 ng/μL RNA (E). Representative experiments are shown.
See also Figures S1 and S2.
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4. Figure 2
An RNA Pseudoknot Is Essential for Activation of PKR and Splicing
(A–C) Stem P2 is essential for the activation of PKR and splicing. Mutated bases are in red and blue. PKR activation (68 kDa) by WT or mutant 2-APRE RNA (0.05 and 0.15 ng/μL) was assayed using rabbit reticulocyte ribosomal fraction (top) and rPKR (bottom); LI is shown (A). BHK-21 cells were transfected with TNF-β gene vector lacking a 2-APRE (TNF-β) or harboring WT or mutant 2-APRE in the 3′ UTR. Splicing efficiency was determined at intervals (error bars, SEM; n = 3) (B). Activation of PKR (LI) is from (A); splicing efficiency at 25 hr is from (B); secreted TNF-β in the experiment of (B) was quantitated at 48 hr post-transfection (error bars, SEM; n = 3) (C).
(D and E) Stem P1 is essential for the activation of PKR and splicing. Activation of rPKR by WT or mutant 2-APRE RNA (0.05 and 0.1 ng/μL) (D). BHK-21 cells were transfected with TNF-β gene vectors. Splicing efficiency was determined at 48 hr; activation of PKR is from (D) (error bars, SEM; n = 3) (E).
See also Figures S3 and S4.
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5. Figure 3
TNF-α 2-APRE Function Relies on Base Pairs Adjoining Pseudoknot Stem P1 and within P2
(A) In the 2-APRE secondary structure, mutated base pairs are encircled.
(B and C) The G13·U37 wobble pair. Mutated bases are marked in red. Activation of rPKR by WT or mutant RNA was assayed (B). BHK-21 cells were transfected with TNF-β gene vector having the WT or G13C 2-APRE inserted into its 3′ UTR; splicing efficiency and secreted TNF-β were determined at 48 hr post-transfection. PKR activation is from (B) (error bars, SEM; n = 3) (C).
(D and E) Mutational analysis of the 5′-proximal base pair (D) and 3′-proximal base pair (E) at the helical junctions in stem P2. Activation of rPKR was assayed.
(F) Stem-loop S3. The Δ(84–95) deletion mutant lacks S3. Activation of rPKR was assayed.
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6. Figure 4
TNF-α 2-APRE Structure and Function Are Conserved from Teleost Fish to Humans
(A) Secondary structure of Homo sapiens 2-APRE (GenBank: X02910).
(B) Three-dimensional model of human 2-APRE RNA, created as described in the Experimental Procedures. Domains are colored as in (A), with the 5′ end magenta.
(C) Secondary structure of Psetta maxima 2-APRE (GenBank: FJ654645), based on multispecies structural alignment. Nucleotide identity with the human sequence is shown in purple.
(D) Three-dimensional model of turbot 2-APRE RNA. To facilitate comparison, turbot domains are colored as in (B).
(E) Activation of rPKR (100 ng per lane) by human and turbot 2-APRE RNA. LI, labeling intensity of PKR band in autoradiogram (percentage of human for each RNA concentration).
(F) Turbot 2-APRE functions in splicing. HEK293T cells were transfected with TNF-β gene vector lacking a 2-APRE (TNF-β) or harboring turbot or human 2-APRE in the 3′ UTR. Splicing efficiency was determined at intervals (error bars, SEM; n = 4).
See also Figure S5 and Movie S1.
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7. Figure 5
PKR-Dependent TNF mRNA Splicing Requires eIF2α Phosphorylation
(A) eIF2αS51A abrogates splicing enhancement by the 2-APRE. BHK-21 cells were co-transfected with TNF-β or TNF-β/2-APRE expression vector and an equal amount of vector expressing WT eIF2α, eIF2αS51A, or empty vector (control). Splicing efficiency was assayed (error bars, SEM; n = 3).
(B) eIF2αS51A expression inhibits splicing of TNF-β mRNA. BHK-21 cells were co-transfected with TNF-β and an equal amount of vector expressing eIF2αS51A or empty vector (control). Splicing efficiency was assayed (error bars, SEM; n = 3).
(C) Expression of phosphomimetic eIF2αS51D does not affect splicing efficiency. BHK-21 cells were co-transfected with TNF-β or TNF-β/2-APRE expression vector and an equal amount of vector expressing eIF2αS51D or empty vector (control). Splicing efficiency was assayed (error bars, SEM; n = 3).
(D) eIF2αS51A, but not eIF2αS51D, inhibits eIF2α phosphorylation by activated PKR. Activation of rPKR (0.15 μg) by 2-APRE RNA (0.1 ng/μL) was assayed in the absence (control) or presence of WT recombinant eIF2α (0.25 μg), mutant eIF2α as indicated, and labeled ATP; phosphorylation of WT eIF2α band (P-eIF2α) is quantitated.
(E and F) Blocking eIF2α dephosphorylation compensates for lack of an RNA activator of PKR in splicing. Western blots show phosphorylated and total eIF2α in BHK-21 cells in response to salubrinal (Sal); a representative of three independent experiments is shown. Ratio of band intensity of phosphorylated over total eIF2α is plotted (error bars, SEM) (E). Splicing efficiency of TNF-β and TNF-β/2-APRE mRNA in BHK-21 cells was assayed in the presence of salubrinal (15 μM) or DMSO (control) (error bars, SEM; n = 3) (F).
See also Figure S6.
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8. Figure 6
TNF mRNA Splicing in Human PBMCs Is Regulated by eIF2α Phosphorylation
(A and B) TNF-α mRNA splicing in PBMCs is enhanced by elevated phospho-eIF2α levels. Splicing efficiency of TNF-α mRNA was assayed by real-time qPCR upon exposure of whole PBMCs to 10 μM PMA, 15 μM salubrinal (Sal), both, or dimethylsulfoxide (none) (error bars, SEM; n = 3) (A). Western blots show phosphorylated and total eIF2α upon induction; a representative of three independent experiments is shown and the ratio of phosphorylated over total eIF2α is plotted (error bars, SEM) (B).
(C) eIF2α phosphorylation strongly inhibits TNF-α protein induction in PBMCs. TNF-α was assayed upon exposure of PBMCs to PMA, Sal, both, or none (error bars, SEM; n = 3).
(D) Salubrinal stimulates TNF-β mRNA splicing in PBMCs. Splicing efficiency of TNF-β mRNA was assayed as in (A) (error bars, SEM; n = 3).
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9. Figure 7
The RNA Activator of PKR in TNF-α Pre-mRNA Upregulates Splicing via eIF2α Phosphorylation
The parallel helix structure of the 2-APRE generated by the pseudoknot facilitates PKR dimerization and activation, providing a molecular basis for the powerful ability of this short intragenic RNA element to activate PKR, inducing eIF2α phosphorylation, both prerequisite to efficient splicing. This distinct function of eIF2α phosphorylation links splicing to the integrated stress response.
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