DXO hydrolyzes only capped RNAs without a 2’-<i>O</i>-methylation.

<p>(A) The RNA 5’ cap structure is composed of a guanosine (blue) linked to the RNA (black) through a 5’-5’ triphosphate bridge. The subsequent N7-methylation of the guanosine (magenta) confers a positive charge to the cap structure. Additional 2’-<i>O</i>-methylations (orange) can be found on the first few nucleotides. (B) Nomenclature of the different cap structures. (C) Aliquots (2μg) of the purified preparations of DXO and mutant DXO protein (D236A/E253A) were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized with Coomassie Blue Dye. The positions and sizes (in kDa) of the size markers are indicated on the left. (D) RNAs harbouring different cap structures were transcribed and capped (incorporation of [α-³²P]GTP) <i>in vitro</i>. They were then subjected to degradation by different enzymes, and reaction products were separated by thin layer chromatography. Lanes 1–4 show reaction products after treatment of differently capped RNAs with Nuclease P1. Degradation products after incubation of differently capped RNAs with purified DXO are shown in lanes 5–12. The origin of spotting and dinucleotide identities are listed on the left. NOTE: During the preparation of differently capped RNAs, only approximately 30% of GpppN-RNA was methylated to form GpppN_m-RNA (lanes 2,7–8), resulting in a mixture of GpppN-RNA and GpppN_m-RNA. Degradation products observed in lane 8 are due to the degradation of GpppN-RNA.</p

2'-<i>O</i>-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO

<div><p>The 5' RNA cap structure (^m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-<i>O</i> position of the first transcribed nucleotide (^m7GpppN_mpRNA) and sometimes on the adjoining nucleotide (^m7GpppN_mpN_mpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5’ ends such as pRNA, pppRNA, GpppRNA, and surprisingly, ^m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of ^m7GpppRNAs by DXO. However, the critical role of the 2’-<i>O</i>-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2’-<i>O</i>-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2’-<i>O</i>-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs.</p></div

Model of DXO activity on RNA.

<p>DXO removes incomplete cap structures such as capG (right) and cap0 (middle) and degrades the resulting uncapped mRNA, whereas RNAs harboring a cap1 structure (left) are unaffected. RNAs with capG and cap0 structures can be either capping intermediates or non-self RNAs.</p

The presence of a 2’-<i>O</i>-methylation blocks the exoribonuclease activity of DXO.

<p>(A) The exoribonuclease activity of DXO toward different substrates was studied. 2μM of DXO were incubated with 100nM of the 30‐nt 3′‐radiolabelled RNA substrate harbouring either no 2’-<i>O</i>-methylation, a 2’-<i>O</i>-methylation on the first nucleotide or a 2’-<i>O</i>-methylation on the 16^th nucleotide. The reactions were incubated at 37°C for 0 to 64 minutes before being stopped by adding 100mM EDTA. Products were separated on a 20% denaturing polyacrylamide gel. (B) To ensure that the observed exoribonuclease activity is specific to the DXO protein, a catalytically inactive mutant (D236A-E253A) was used in an exoribonuclease assay with 5’ monophosphorylated RNA. Wild-type DXO readily degrades this RNA substrate, whereas almost no cleavage products are observed with the inactive mutant.</p

DXO contains a functional NLS as shown by site-directed mutagenesis and immunofluorescence.

<p>(A) A schematic representation of a classical bipartite NLS consensus sequence and the corresponding sequence at the N-terminal end of DXO. (B) HeLa cells were transfected with pcDNA3.1+/DXO and pcDNA3.1+/DXO-K7A-R8A (mutations in the NLS) using Lipofectamine 2000. DXO localization was monitored 48 hours post-transfection by immunofluorescence using a rabbit polyclonal DXO antibody and a polyclonal anti-rabbit antibody coupled to Alexa Fluor 488. Fluorescent images were gathered using an epifluorescence microscope with a 60x objective. Exposure times were identical in all three conditions.</p

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

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

Comparison between EBV-negative and EBV-associated gastric cancer.

<p><b>(A)</b> Heatmap representation of isoform ratios (PSI values) for EBV-negative gastric adenocarcinomas tissues. EBV-negative gastric adenocarcinomas tissues (TNoV) are shown in blue, and the comparative healthy tissues are shown in green (NNoV). <b>(B)</b> Comparison of the cellular genes with dysregulated ASEs between EBVaGC and EBV-negative GC tissues. <b>(C)</b> The list displays common differentially spliced transcripts for both EBV-negative and EBVaGC with the corresponding Delta PSI values, the associated gene expression (in Log₂), and the related biological processes.</p

Identification of alternative splicing events in gastric cancer.

<p><b>(A)</b> Overview of the strategy used to identify the changes in alternative splicing in GC. <b>(B)</b> Classification of the TCGA RNA sequencing data for GC and healthy tissues. <b>(C)</b> The splicing events list for both EBV-negative gastric adenocarcinomas (TNoV, Tumors, no virus) and EBV-associated gastric carcinomas (EBVaGC, TEBV, Tumors with EBV) compared the global splicing patterns with normal tissues (NNoV, Normal tissues, no virus) was filtered to keep only significant ASEs.</p