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Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner.

ABSTRACT: The herpes simplex virus (HSV) infected cell culture polypeptide 27 (ICP27) protein is essential for virus infection of cells. Recent studies suggested that ICP27 inhibits splicing in a gene-specific manner via an unknown mechanism. Here, RNA-sequencing revealed that ICP27 not only inhibits splicing of certain introns in <1% of cellular genes, but also can promote use of alternative 5' splice sites. In addition, ICP27 induced expression of pre-mRNAs prematurely cleaved and polyadenylated from cryptic polyadenylation signals (PAS) located in intron 1 or 2 of ?1% of cellular genes. These previously undescribed prematurely cleaved and polyadenylated pre-mRNAs, some of which contain novel ORFs, were typically intronless, <2 Kb in length, expressed early during viral infection, and efficiently exported to cytoplasm. Sequence analysis revealed that ICP27-targeted genes are GC-rich (as are HSV genes), contain cytosine-rich sequences near the 5' splice site, and have suboptimal splice sites in the impacted intron, suggesting that a common mechanism is shared between ICP27-mediated alternative polyadenylation and splicing. Optimization of splice site sequences or mutation of nearby cytosines eliminated ICP27-mediated splicing inhibition, and introduction of C-rich sequences to an ICP27-insensitive splicing reporter conferred this phenotype, supporting the inference that specific gene sequences confer susceptibility to ICP27. Although HSV is the first virus and ICP27 is the first viral protein shown to activate cryptic PASs in introns, we suspect that other viruses and cellular genes also encode this function.

SUBMITTER: Tang S

PROVIDER: S-EPMC5087043 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner.

Tang Shuang S Patel Amita A Krause Philip R PR

Proceedings of the National Academy of Sciences of the United States of America 20161010 43

The herpes simplex virus (HSV) infected cell culture polypeptide 27 (ICP27) protein is essential for virus infection of cells. Recent studies suggested that ICP27 inhibits splicing in a gene-specific manner via an unknown mechanism. Here, RNA-sequencing revealed that ICP27 not only inhibits splicing of certain introns in <1% of cellular genes, but also can promote use of alternative 5' splice sites. In addition, ICP27 induced expression of pre-mRNAs prematurely cleaved and polyadenylated from cr ...[more]

PMID: 27791013

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Project description:Background:In a previous study, we showed that consumption of diets enriched in saturated fatty acids causes changes in alternative splicing of pre-mRNAs encoding a number of proteins in rat skeletal muscle, including the one encoding skeletal muscle Troponin T (Tnnt3). However, whether saturated fatty acids act directly on muscle cells to modulate alternative pre-mRNA splicing was not assessed. Moreover, the signaling pathway through which saturated fatty acids act to promote changes in alternative splicing is unknown. Therefore, the objective of the present study was to characterize the signaling pathway through which saturated fatty acids act to modulate Tnnt3 alternative splicing. Methods:The effects of treatment of L6 myotubes with saturated (palmitate), mono- (oleate), or polyunsaturated (linoleate) fatty acids on alternative splicing of pre-mRNA was assessed using Tnnt3 as a marker gene. Results:Palmitate treatment caused a two-fold change (p?<?0.05) in L6 myotube Tnnt3 alternative splicing whereas treatment with either oleate or linoleate had minimal effects compared to control myotubes. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on Tnnt3 alternative splicing and inhibition of de novo ceramide biosynthesis blocked the palmitate-induced alternative splicing changes. The effects of palmitate and ceramide on Tnnt3 alternative splicing were accompanied by a 40-50% reduction in phosphorylation of Akt on S473. However, inhibition of de novo ceramide biosynthesis did not prevent palmitate-induced Akt dephosphorylation, suggesting that palmitate may act in an Akt-independent manner to modulate Tnnt3 alternative splicing. Instead, pre-treatment with okadaic acid at concentrations that selectively inhibit protein phosphatase 2A (PP2A) blocked both palmitate- and ceramide-induced changes in Tnnt3 alternative splicing, suggesting that palmitate and ceramide act through PP2A to modulate Tnnt3 alternative splicing. Conclusions:Overall, the data show that fatty acid saturation level and ceramides are important factors modulating alternative pre-mRNA splicing through activation of PP2A.

Dataset Information

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner.

Publications

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner.

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