BackgroundDysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data.ResultsOur study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control.ConclusionsThis study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3029-z) contains supplementary material, which is available to authorized users.

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

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'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). 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’-O-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’-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2’-O-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.

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.

eukaryotic viruses require strategies, such as RNA cap synthesis, in order to protect, replicate and translate their genomes in eukaryotic hosts. Figure 1. RNA 5'-cap structure. The RNA 5'cap structure is composed of a 7-methylguanosine (blue) linked to the RNA (black) through a 5'-5' triphosphate bridge (blue and black). The N7 methylation of the guanosine (green) confers a positive charge to the cap structure. Additional 2'O-methylation (red) can be found on the first few nucleotides. Conventional and unconventional 5' RNA cap synthesis mechanism Canonical cap synthesis by different virusesThe importance of the cap structure in eukaryote metabolism has resulted in an evolutionary pressure for viruses to adopt a similar cap structure. A series of enzymatic reactions is required to synthesize a cap structure at the 5'-end of RNA. The most pervasive enzymatic pathway, also termed "conventional capping", consists of three sequential enzymatic activities that are required to generate a functional 7-methylguanosine 5'-5'-triphosphate bridged cap structure. As a result of the directional 5' to 3' polymerization of nucleotide triphosphates (NTP) during RNA synthesis, nascent RNA bear at their 5'-end a triphosphate moiety (originating from the initial NTP). This 5'-triphosphate end of the RNA is first converted into a 5'-diphosphate end by hydrolysis of the terminal phosphate, or γ-phosphate, by an RNA triphosphatase (RTPase). This is followed by a two-step reaction catalyzed by an RNA guanylyltransferase (GTase). The enzyme first specifically binds and hydrolyzes a GTP molecule to form a covalent enzyme-GMP intermediate, which then catalyzes the transfer of the GMP moiety onto the 5'-end of a diphosphorylated acceptor RNA (ppRNA) in the second step of GTase reaction. Lastly, an RNA 5′-end Maturation: A Crucial Step in the Replication of Viral Genomes