Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas

<div><p>Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.</p></div

Validation of ASEs dysregulated in infected cells.

<p>(A) Overview of two isoforms encoded by the <i>ABI1</i> gene. Exons are depicted in red and the intervening introns are shown as thin black lines (not to scale). The primers used to detect the ASE by RT-PCR assays are shown in gray and the sizes of the expected amplicons (306 nt and 393 nt) are also indicated. The genomic coordinates of these two representative isoforms are also indicated. (B) Cellular mRNAs isolated from both uninfected and infected cells were analyzed by RT-PCR using specific primers to detect both forms of the modified ASE encoded by the <i>ABI1</i> gene. The amplified products were analyzed by automated chip-based microcapillary electrophoresis. Capillary electropherograms of the PCR reactions are shown. The positions and the amplitude of the detected amplicons are highlighted by red boxes. The positions of the internal markers are also indicated. The data shows the increase in the relative abundance of the short form (306 nt) and a decrease in the abundance of the long form (393 nt) upon viral infection. (C) Correlation between PSI values obtained from RNA-Seq and RT-PCR data. The analysis was performed on 16 selected ASEs (Abi1, Cwc22, Eif4a2, Hnrnpa2b1, Il34, Srsf3, Srsf5, Alkbh1, Cdkn2aip, Cflar, Hif1a, Mdm2, Serbp1, Sfswap, Smc2, Tbp). In all cases, the changes in AS levels detected by RT-PCR and the ones revealed through transcriptome sequencing displayed high levels of correlation (r >0.77). Sanger sequencing was also realized on several ASEs to confirm that RT-PCR reaction is specific and amplifies predicted ASE.</p

Protein families found in the 240 transcripts that are differentially spliced upon viral infection.

<p>Protein families found in the 240 transcripts that are differentially spliced upon viral infection.</p

Global profiling of the cellular alternative splicing landscape and identification of differentially spliced ASEs during virus-host interactions.

<p>(A) Heatmap representation of isoform ratios for cellular transcripts in both infected and uninfected (mock) cells. RNA sequencing was done in triplicate for each condition. The map represents the percent-spliced-in (PSI) values based on isoform expression for the long and the short ASEs (see Materials and methods). Blue indicates high PSI values and red indicates low PSI values. (B) Alternative splicing events (ASEs) in cells infected with reovirus. ASEs were detected and quantified using the percent-spliced-in (PSI) metric. The graph shows an analysis of the difference in PSI values (Delta PSI) of the cellular genes following viral infection. Black triangles indicate Delta PSI values between -10 and 10, red squares indicate Delta PSI values greater than 10 or less than -10 with a Q value under 0.05, and green circles indicate same Delta PSI values but with a Q value above 0.05. (C) Heatmap representation of the 240 ASEs that are differentially spliced upon viral infection. RNA sequencing was done in triplicate for both the uninfected (mock) and infected cells. Blue indicates high absolute PSI values and red indicates low absolute PSI values. (D) PSI distribution of the 40 primary ASEs for which AS is the most significantly altered upon viral infection. The PSI values for the respective ASEs are indicated both for the uninfected and infected cells. Error bars indicate standard deviation.</p

Proteins involved in RNA splicing.

<p>(A) Iris graph displaying the expression profile of proteins involved in RNA splicing. Differences in gene expression levels are shown on a logarithmic color scale (Log2). The expression of only 10 proteins involved in splicing was modulated by more than 2-fold upon infection (indicated by an asterisk). (B) Modifications to the splicing profiles of proteins involved in RNA splicing upon viral infection. Nine splicing factors were differentially spliced following viral infection. The changes in PSI values are indicated in red (negative delta PSI) or green (positive delta PSI).</p

Transcriptomic studies of cells infected with reovirus.

<p>(A) Overview of the strategy used to identify the changes in both the cellular transcriptome and alternative splicing landscape upon reovirus infection. RNA-seq analysis was performed on infected cells at 14 hours post-infection. (B) MA-plot of cellular gene expression levels upon viral infection as compared to uninfected cells. The graph shows the fold-change (FC) in base 2 logarithm between infected and mock cells according to the mean expression of the gene in transcripts per million (TPM, also presented in log₂). A cluster of over-expressed genes during viral infections with high TPM value can be seen on the upper right corner. (C) Gene ontology analyses of the 569 genes for which the expression was the most significantly modified following viral infection. Up- and down-regulated genes were imported into the DAVID gene ontology suite of programs at the NIAID. Ontological functions were determined for biological processes, and background of all detected genes was used.</p

Proteomic analysis of uninfected and reovirus-infected cells.

<p>(A) <i>LRRFIP1</i> coding exon structure and localization of peptides detected for this gene. Two long isoform transcripts (NM_001111312; NM_008515) and one short isoform transcript (NM_001111311) are also represented. Other short (BC144955; AK044174) and long (BC145642) isoforms analyzed in the RNA-Seq process were omitted. (B) Transcript-specific peptides detected for the long/short transcript. The intensities of peptide detection for both uninfected (mock) and infected cells in the cytoplasm/nucleus fractions are displayed as color gradation (Cytoplasm: grey = weakly detected, blue = strongly detected, white = undetected; Nucleus: grey: lightly detected, red = strongly detected; white = undetected). For Lrrfip1, two peptides (A and B) are presented. (C) Fold enrichment of reovirus proteins in nuclear fraction over the cytoplasmic one. Proteins μ2 (74.5x), λ3 (26.5x) and σNS (15.4x) were found to be above the background level of other viral proteins.</p

Characterization of the ASEs that are modified upon viral infection.

<p>(A) The percentage of splicing profiles found in cells and in the 240 differentially spliced transcripts are presented. Schematic representations of the various splicing profiles are also indicated as well as the statistical significance (chi-square with Yates' correction). (B) Distribution of the number of ASEs per gene. The vast majority of the ASEs that are modified upon viral infection were found at a level of one splicing event per gene. (C) Gene expression levels of the 240 differentially spliced transcripts. The graph displays both the modifications in gene expression and alternative splicing of the 240 ASEs that are differentially spliced upon viral infection. The variations in gene expression are presented in a logarithmic scale (Log₂).</p

Involvement of EBNA1 in alternative splicing.

<p><b>(A)</b> Immunoblotting analysis using anti-HA antibody for the detection of EBNA1-HA-FLAG protein in cell lysates from a stable HEK293T cell line expressing EBNA1. Control HEK293T cells (T(-)) were also used in this assay. <b>(B)</b> List of ASEs common to EBVaGC and EBNA1-expressing cells. <b>(C)</b> Example of a modified ASE following the expression of EBNA1. Overview of the two isoforms encoded by <i>OSBPL9</i> gene. Exons are represented in red and the intervening introns are displayed as thin black lines (not to scale). The primers used to detect the isoforms by RT-PCR assays are presented in gray and the sizes of the expected amplicons are also specified (top panel). RT-PCR reactions were performed on control cells (T(-)) and cells expressing EBNA1 using specific primers to detect both isoforms of the transcripts encoded by the <i>OSBPL9</i> gene. Capillary electrophoresis assays were performed and an image of the detected reaction products is presented (lower panel). The positions of the expected amplicons are shown by arrows. <b>(D)</b> Mass spectrometry analysis of nuclear proteins interacting with EBNA1. The average ratios (MS/MS counts) of the EBNA1 affinity purification-mass spectrometry experiments were plotted versus the total intensities. <b>(E)</b> Validation of the interaction between EBNA1 and splicing factor hnRNP H1. Nuclear extracts were immunoprecipitated with anti-HA. The extracts (input) and immunoprecipitates (IP-EBNA1) were analyzed by immunoblotting and probed with the indicated antibodies.</p

Modifications to AS of 96 transcripts in response to knockdown of specific splicing factors with siRNAs.

<p>Using specific siRNAs, eighteen splicing factors (U2AF2, U2AF1, SYNCRIP, SFRS9, SFRS6, SFRS2, NOVA1, KHSRP, KHDRBS1, HNRPU, HNRPR, HNRPM, HNRPK, HNRPH1, HNRPF, HNRPD, HNRPC, and HNRPA1) were individually knocked-down in various cell lines to evaluate their implication in splicing of 96 different transcripts. Asterisks (top) indicate transcripts for which AS was altered in GC. Individual knockdowns and ASEs are presented to indicate which knockdowns produced a shift in AS in various cell lines (PC-3, SKOV3, NIH:OVCAR-3, MDA-MB-231, MCF7). Each individual column represents a different knockdown performed with specific siRNAs. The changes in PSI values are indicated. The map displays the changes in PSI values in a color-coded scale. White areas indicate no shifts.</p