Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators - PubMed (original) (raw)

doi: 10.1128/MCB.06130-11. Epub 2011 Dec 27.

Alexandre Cloutier, Johanne Toutant, Lulzim Shkreta, Philippe Thibault, Mathieu Durand, Daniel Garneau, Daniel Gendron, Elvy Lapointe, Sonia Couture, Hervé Le Hir, Roscoe Klinck, Sherif Abou Elela, Panagiotis Prinos, Benoit Chabot

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Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators

Laetitia Michelle et al. Mol Cell Biol. 2012 Mar.

Abstract

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

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Figures

Fig 1

Fig 1

EJC components modulate the ratio of Bcl-x splice forms in different cell lines in a NMD-independent manner. (A) Diagram representing the structure of the Bcl-x gene and its splice variants. (B) Impact of depleting Y14 and RNPS1 in MDA-MB-231, PC-3, 293, and HeLa cells on the relative abundance of Bcl-x mRNA splice forms. The percentage of Bcl-xS is indicated below each lane. (C) RNAi assays were extended to other core and auxiliary EJC components. Two different siRNAs per gene (black and white bars) were transfected in 293 and HeLa cells. Seventy-two hours later, the impact of these depletions on the Bcl-x splicing profile was investigated. Experiments were done in triplicates, and standard deviations are shown. Western blot analyses are shown on the right. (D) The samples generated for panel C were investigated for changes in the expression of TIMM8B and ILK splice forms. Isoforms containing a premature stop codon (PTC) are indicated. (E) HeLa cells transfected with siRNAs targeting Upf1, Upf2, and Upf3b were investigated for changes in the expression of Bcl-x, TIMMB8, ILK, and CPNE2 splice variants.

Fig 2

Fig 2

EJC components require distinct _cis_-acting elements to modulate Bcl-x pre-mRNA splicing. (A) Structures of the Bcl-x minigenes. X2 differs from X2.13 by the presence of the SB1 element (underlined, 361 nt) located at the 5′ end of exon 2 (Ex2) (56). Derivatives of X2.13 lacking the 50-nucleotide-long B1 or the 77-nucleotide-long B2 element are also shown (23). P, promoter. (B) RT-PCR analysis of minigene expression was carried out in 293 cells treated with siRNAs targeting RNPS1, Y14, eIF4A3, and PYM, followed by transfection of the Bcl-x minigenes. (C) RT-PCR analysis of Bcl-x expression in 293 cells was carried out after cotransfecting FLAG-RNPS1, FLAG-Y14, and FLAG-PYM constructs with the Bcl-x minigenes. (D) Diagram of the SB1 element and of the various 40-bp (Δ9-12), 70-bp (Δ13-19), and 10-nt (Δ13 to Δ19) deletion mutants tested. Plasmid transfection was performed in 293 cells in the absence or presence of the FLAG-RNPS1 plasmid. To illustrate the impact of the mutant and that of RNPS1 coexpression, Bcl-x splicing is expressed as the percentage of the Bcl-xS isoform. s18 represents a substitution mutation with the sequence 5′-CTTCTCTTGT-3′. (E) RT-PCR analysis of Bcl-x alternative splicing in 293 cells transfected with an siRNA targeting Y14 and then with minigene X2.13 and versions lacking the B1 or B2 element. RT-PCR was performed to amplify the Bcl-x isoforms produced from the minigenes. Bar graphs in panels B, C, and E show the positive or negative difference in the production of Bcl-xS in cells where the expression of specific EJC components was increased or decreased relative to that in mock-treated cells.

Fig 3

Fig 3

EJC components associate with the Bcl-x pre-mRNA and modulate its alternative splicing. (A) The structures of the Bcl-x and ILK pre-mRNA are diagrammed, and the positions of the primers used to amplify intron-containing (A-B pairs) or intron-lacking (A-C pairs) transcripts are shown. RT-PCR analysis was carried out after immunoprecipitating the RNA using a variety of antibodies in 293 total cell extracts. Sepharose-protein A beads linked to antibodies against the eIF4A3, Y14, Magoh, RNPS1, and Sm proteins (Y12) and PABP were added to extracts. Bound RNA was recovered after extensive washing and treatment with proteinase K and was analyzed by RT-PCR using primers specific to Bcl-x or the PTC-containing ILK splicing unit. (B) Following incubation of Bcl-x and control hnRNP A1-derived (45) _in vitro_-transcribed RNA in a HeLa nuclear extract, labeled RNA was immunoprecipitated with anti-eIF4A3, anti-Y14, and anti-Magoh antibodies. Incubation was performed in splicing mixtures depleted of ATP. The amount of immunoprecipitated RNA was quantitated and plotted to show the fold difference of precipitated material relative to control beads, with a value of 1 indicating no difference. (C) In vitro splicing assay in HeLa nuclear extracts. Splicing assays were performed with 2 fmol of the Bcl-x pre-mRNA S2.13 (13) or RNA 45 (derived from hnRNP A1 [45]) in the presence of increasing amounts of His-tagged recombinant eIF4A3 or Magoh-Y14ΔN (3). (D) Diagram of the B2 element and the subregions that define the ΔB2G, ΔB2.2, and ΔB2.1 deletion mutants. (E) In vitro splicing assays performed as for panel C using the recombinant Y14-Magoh mixture (left panel) or the recombinant eIF4A3 (right panel). The Bcl-x pre-mRNAs tested are indicated, as well as the final concentrations of recombinant proteins. Bar graphs show difference in the production of Bcl-xS in Y14-Magoh- or eIF4A3-supplemented extracts relative to extracts receiving only buffer D.

Fig 4

Fig 4

eIF4A3 interacts directly with the Bcl-x pre-mRNA via the B2 element. (A) Diagram of the alternative exonic region located between the alternative Bcl-x 5′ splice sites. The B2 element is depicted with its subregions B2G, B2.2, and B2.1. The segment designated WT RNA represents the _in vitro_-transcribed RNA used for panels C and D. (B) Following the incubation of the various Bcl-x transcripts in a HeLa nuclear extract, labeled RNA was immunoprecipitated with anti-eIF4A3. The percentage of immunoprecipitated labeled RNA (relative to input and subtracted from that for a mock immunoprecipitation performed with each pre-mRNA) is plotted. (C) Cross-linking assays were performed in the presence of labeled WT or ΔB2 transcripts incubated for 30 min on ice with recombinant His-eIF4A3 or a HeLa nuclear extract, followed by UV irradiation and immunoprecipitation with anti-eIF4A3. Samples were fractionated on a 10% polyacrylamide-SDS gel. The left panel displays a Coomassie blue-stained gel of our purified His-eIF4A3 protein. (D) Labeled WT or ΔB2 transcripts were incubated for 25 min at 30°C in the presence (+) or the absence (−) of 1 μg recombinant protein His-eIF4A3. Complexes were separated on a native 4.5% acrylamide gel.

Fig 5

Fig 5

EJC components control a network of splicing events linked to apoptosis. (A) RT-PCR analysis monitoring the alternative splicing of 54 units following the knockdown of EJC components and Upf1 in HeLa cells. Total RNA was extracted from HeLa cells at 72 h after transfection with siRNAs. The colored boxes represent significant shifts (minimum 10%) in the percent splicing index (PSI). The “PTC” column indicates the presence of a premature termination codon introduced by alternative splicing by either inclusion or exclusion. If the PTC is located more than 50 nucleotides upstream of an exon-exon junction, it is predicted to be a target for NMD and hence the event is indicated as a black box (NMD substrate). If the PTC is located within the 50 nucleotides upstream of the exon-exon junction or downstream of the last exonic junction, the transcript is assumed to escape NMD (NMD immune) and is indicated as a gray box. (B) Venn diagram showing the numbers of transcripts regulated by Y14, eIF4A3, and UPF1. Transcripts are considered coregulated by two or three of these proteins only if the splicing ratio shifts in the same direction upon depletion of the proteins. (C) Annexin V assay in HeLa cells transfected with siRNAs targeting EJC components. Cells were immunostained for annexin V (red), and nuclei were labeled with Hoechst stain (blue) (69). The percentage of apoptotic cells was quantitated (bottom graph) upon transfection of siRNAs targeting eIF4A3, Y14, and RNPS1 (2 siRNAs each) and compared to that for various controls (Lipofectamine alone, negative-control siRNAs targeting KITLG and cyclin D1, and a positive-control siRNA targeting KIF11 [71]).

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