When SUMO met splicing

<p>Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.</p

Pharmacological modulation of Akt with Akt-IV activates all three UPR branches: PERK responds first.

<p>(<b>A</b>) When activated, IRE1 processes <i>Xbp1</i> mRNA by a non-conventional cytoplasmic splicing reaction, changing <i>Xbp1</i> open reading frame. HEK293T cells were treated with Akt-IV (10 µM), Akt-VIII (5 µM) or LY294002 (20 µM) for the indicated times. <i>Xbp1</i> mRNA splicing was detected by RT-PCR. <i>Xbp1s</i>: spliced form (activated IRE1); <i>Xbp1u</i>: unspliced form (inactive IRE1). (<b>B</b>) When activated, ATF6 translocates to the Golgi apparatus where it is cleaved to release a fragment that enters the nucleus where it functions as a transcription factor. HEK293 cells were transfected with ATF6-Flag plasmid and 24 h later they were treated with Akt-IV (10 µM), Akt-VIII (5 µM), LY294002 (20 µM), or thapsigargin (Tg; 100 nM) for the indicated times. Western blots (WB) using antibodies against FLAG and actin are shown for every case (B, upper panel). ATF6: uncleaved protein; ATF6f: cleaved form. (<b>C</b>) HEK293T cells were transfected with a plasmid that expresses the YFP-NLS-mATF6short reporter (top, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069668#pone.0069668.s001" target="_blank">Fig. S1A</a> for details). Forty-eight hours post-transfection cells were treated for the indicated times with Akt-IV and then fixed, DNA was stained with DAPI and cells were imaged (lower panel); Yellow, YFP-ATF6; Blue, DNA; scale bar, 5 µm. For all cases cells treated with DMSO were used as a control (Control). (<b>D</b>) When activated, PERK is autophosphorylated at multiple residues and activated to phosphorylate eIF2α. HEK293T cells were treated with Akt-IV (10 µM), Akt-VIII (5 µM) or LY294002 (20 µM) for the indicated times. Protein extracts were analyzed by WB using the indicated antibodies. Data in the plot corresponds to ratio of phosphorylated total abundance of each of the indicated proteins (normalized to the initial value) in cells treated with the indicated drugs for different times. Error bars correspond to the standard error of three independent experiments. (<b>E</b>) HEK293T cells were treated for 5 h with Akt-IV. peIF2α abundance was detected by immunofluorescence. Green, peIF2α; Blue, DNA; scale bar, 5 µm. Data are representative of at least three independent experiments.</p

Akt-IV stimulation of eIF2α phosphorylation is Akt- and PERK- dependent but PI3K-independent.

<p>(<b>A</b>) MEF WT or Akt DKO cells were treated with Akt-IV (IV; 10 µM) for the indicated times. <i>Xbp1</i> mRNA splicing was detected by RT-PCR. <i>Xbp1s</i>: spliced form (activated IRE1); <i>Xbp1u</i>: unspliced form (inactive IRE1). (<b>B</b>) MEF WT or Akt DKO cells were treated with Akt-IV (IV; 10 µM) for 1 h. Protein extracts were analyzed by WB using the indicated antibodies. The fold change in peIF2α/eIF2α ratio induced by Akt-IV was quantified for three independent experiments. On average, this fold change was reduced to 15% of the original effect in MEF Akt DKO cells compared to WT cells (11.0 vs 2.6). (<b>C</b>) MEF WT cells were transfected with HA-Akt or HA-Akt KM plasmids. Forty-eight hours post-transfection cells were treated with DMSO (C) or with Akt-IV (IV; 10 µM) for 1 h. A GFP expressing plasmid was used as a transfection control. Protein extracts were analyzed by WB using the indicated antibodies. (D) MEFs WT or PERK−/− were treated with Akt-IV (IV; 10 µM) for the indicated times. Protein extracts were analyzed by WB using the indicated antibodies. (<b>E</b>) HEK293T cells were pretreated with DMSO (C) or LY294002 (LY; 20 µM) for 30 min and then treated for 1 h with DMSO, or Akt-IV (without removing the corresponding pre-treatment). Protein extracts were analyzed by WB using the indicated antibodies. Data are representative of at least three independent experiments.</p

A physiological link between Akt and PERK/eIF2α.

<p>(A) WT or Akt DKO MEF cells were subjected to normoxia (C) or hypoxia (0.1%±0.1 O₂) (H) for the indicated times. Protein extracts were analyzed by WB using the indicated antibodies. The fold change in peIF2α/eIF2α ratio induced by hypoxia was quantified for two independent experiments. On average, this fold change was reduced to 0, 40 or 60% of the original effect in MEF Akt DKO cells compared to WT cells (1 h, 2 h and 4 h, respectively). (B) A model summarizing our results. Akt-IV (or other stimuli, such as hypoxia) targets an unknown kinase, possibly PDK1, triggering apoptotic cell blebbing and activating Akt in a PI3K-independent manner (1). Subsequently, UPR is activated (2). Akt presence and activity are necessary for eIF2α phosphorylation due to the existence of a connection between Akt and PERK/eIF2α signaling pathways. IRE1 (4) and ATF6 (5) are activated are later times. At the end, activation of IRE1 and PERK and dephosphorylation of Akt and GSK3β are associated with cell death (6).</p

Akt-IV induces cell blebbing and a UPR dependent cell death.

<p>(<b>A</b>) HEK293T cells were treated with DMSO (C), 100 µM of the caspase inhibitor ZVAD, 10 µM Akt-IV (IV) or both. PERK mobility, eIF2α phosphorylation, Akt phosphorylation on Ser473, caspase 3 cleavage and PARP cleavage were detected by WB. (<b>B</b>) Transmission images of HeLa cells treated for 15 min with DMSO (15 min) or with Akt-IV (10 µM), with blebs indicated. Bleb formation was clearly observed in HeLa and MEF cells but could not be detected in HEK293T cells. (<b>C</b>) YFP channel images of HeLa cells transfected with pAkt1-YFP plasmid and then treated for the indicated times with Akt-IV (10 µM). Akt1-YFP can be detected in blebs after 15 min of treatment. (<b>D</b>) HeLa cells were treated for 15 min with Akt-IV. Cells were fixed and immunostained against pAkt substrate/Alexa Fluor® 488 and total eIF2α/Alexa Fluor® 594. Green, pAkt substrate; Red, eIF2α; Blue, DNA. scale bar, 5 µm. (<b>E</b>) HeLa cells were treated for different times with Akt-IV (10 µM) and then cells were fixed and immunostained for pAkt substrate/Alexa Fluor® 488; scale bar, 5 µm. (<b>F</b>) MEF WT, IRE1^−/− or PERK^−/− were treated with Akt-IV (10 µM) for 12 h. Cell viability was measured by flow cytometry using propidium iodide. Data are representative of at least three independent experiments.</p

Strategy.

<p>(<b>A</b>) Chemical structure of the compounds targeting the PI3K/Akt pathway used in this study. (<b>B</b>) Scheme of Akt signaling pathway, which regulates cell survival, showing the point of action of the drugs shown in <b>A</b>. Akt phosphorylation and activation result from its recruitment to PIP₃ at plasma membrane, after which it exerts cytoplasmic and nuclear functions. Accumulation of PIP₃ classically follows ligand (L) binding to tyrosin kinase cell-surface receptors (RTK), adapter proteins (AP) recruitment to RTK and finally, PI3K activation to phosphorylate PIP₂ to PIP₃. While Akt can be activated by the UPR it is not known if Akt can also regulate the UPR.</p

Silencing U5 snRNP components enhances DENV replication.

<p>(A) Reporter DENV replication in A549 cells transfected with siRNA directed to spliceosomal proteins (CD2BP2, DDX23, SNRNP40, PRPF8, EFTUD2, SNRNP200 or SF3A2). An infectious DENV carrying the luciferase gene was used. Three controls were included: a non-related siRNA (NR, black bar), siRNAs directed to Renilla luciferase (control for inhibition, red bar) and siRNA directed to STAT2 (control of NS5 binder with antiviral activity, green bar). Results are representative of three independent experiments (duplicates, mean ± SD). At the bottom, immunoblots indicate the levels of silenced proteins. (B) DENV replication in A549 cells transfected with a non-related siRNA (NR), or siRNAs directed to CD2BP2, DDX23 or EFTUD2, measured by quantitative real time PCR (mean ± SD) at 10hpi for EFTUD2 and 24hpi for CD2BP2 and DDX23. (C) Induced levels of mRNAs corresponding to RIGI, ISG15 and IL8 in A549 cells infected with NDV, which were previously silenced with siRNAs directed to CD2BP2 or EFTUD2 as indicated for each case. RNA levels were measured by quantitative real time PCR (mean ± SD). On the right, immunoblots indicate the levels of silenced proteins and loading controls.</p

DENV infection modifies the splicing landscape of host cells.

<p>(A) Strategy to detect and quantify alternative splicing isoforms in mock infected or DENV infected cells is depicted on the left. Representative autoradiographs and quantification of the inclusion/exclusion ratio for two alternative exon cassette events are shown (duplicates, mean ± SD). (B) Fold-change of inclusion/exclusion ratios for DENV or mock infected cells. A battery of endogenous alternative exon-cassette events (duplicates, mean ± SD) in A549 and Huh7 cells is shown. (C) DENV-induced changes in the splicing pattern of IKKε and MxA mRNAs. Quantification of alternative splicing events is shown for each case.</p

Decreased splicing efficiency in DENV infected cells.

<p>(A) Schematic representation of splicing analysis. The four splicing events analyzed are shown: Intron retention (IR), Exon skipping (ES), Alternative splice site donor/acceptor (Alt5’SS and Alt3’-SS). Constitutive exon and alternative 5’ and 3’SS regions are shown in grey and intron regions in pink. For intron retention the information of the junctions (E1-I, IE-2 and E1E2) was used to calculate the PIR metric. For ES and AltSS the PSI metric was used (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005841#sec012" target="_blank">Materials and methods</a>). (B) Data of splicing analysis of DENV or mock infected cells. On the upper panels, total amount of bins analyzed and altered splicing events for each time point post infection (24 and 36 hours). The percentage of altered events (ES, ALtSS and IR) is shown in pie charts. On the lower panel, increase or decrease retention of introns for each time point is shown.</p

NS5 alone interferes with alternative splicing.

<p>(A) NS5 shows a dose dependent effect on the splicing reporter minigenes CFTR, EDI, and Bclx. Radiolabeled amplification products corresponding to different splicing isoforms derived from the indicated minigenes are shown. (B) Quantitative analysis of the changes induced by NS5 (0.9 μg of plasmid). (C) Expression of NS5 mutants with impaired methyltransferase (Mut-MTase) or polymerase (Mut-RdRp) enzymatic activity also alters alternative splicing of reporter genes.</p