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. 2016 Sep 6;11(9):e0161914.
doi: 10.1371/journal.pone.0161914. eCollection 2016.

Global Profiling of the Cellular Alternative RNA Splicing Landscape During Virus-Host Interactions

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Free PMC article

Global Profiling of the Cellular Alternative RNA Splicing Landscape During Virus-Host Interactions

Simon Boudreault et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Transcriptomic studies of cells infected with reovirus.
(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.
Fig 2
Fig 2. Global profiling of the cellular alternative splicing landscape and identification of differentially spliced ASEs during virus-host interactions.
(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.
Fig 3
Fig 3. Validation of ASEs dysregulated in infected cells.
(A) Overview of two isoforms encoded by the ABI1 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 ABI1 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.
Fig 4
Fig 4. Characterization of the ASEs that are modified upon viral infection.
(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).
Fig 5
Fig 5. Proteomic analysis of uninfected and reovirus-infected cells.
(A) LRRFIP1 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.
Fig 6
Fig 6. Proteins involved in RNA splicing.
(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).

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References

    1. McCormick AL, Mocarski ES Jr. Viral modulation of the host response to infection In: Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, et al. , editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007. Available: http://www.ncbi.nlm.nih.gov/books/NBK47417/.
    1. Nagy PD, Pogany J. The dependence of viral RNA replication on co-opted host factors. Nat Rev Microbiol. 2012;10(2):137–49. - PMC - PubMed
    1. Walsh D, Mathews MB, Mohr I. Tinkering with translation: Protein synthesis in virus-infected cells. Cold Spring Harb Perspect Biol. 2013;5(1):a012351 10.1101/cshperspect.a012351 - DOI - PMC - PubMed
    1. Hao L, Sakurai A, Watanabe T, Sorensen E, Nidom CA, Newton MA, et al. Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature. 2008;454(7206):890–3. 10.1038/nature07151 - DOI - PMC - PubMed
    1. Kushner DB, Lindenbach BD, Grdzelishvili VZ, Noueiry AO, Paul SM, Ahlquist P. Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc Natl Acad Sci. 2003;100(26):15764–9. - PMC - PubMed

Grant support

This work was supported by grants from the Centre de recherche du Centre hospitalier universitaire de Sherbrooke (MB and JPP), the Faculté de médecine et des sciences de la santé de l’Université de Sherbrooke (MB and JPP), and the Natural Sciences and Engineering Research Council of Canada (GL). JPP holds the Université de Sherbrooke Research Chair in Structure and Genomic RNA. MB is a Chercheur-boursier Sénior from the Fonds de recherche du Québec-Santé (FRQ-S), and SB holds graduate training awards from the FRQ-S and Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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