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Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene.

ABSTRACT: We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IVPDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 microM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 microM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.

SUBMITTER: Bolger GB

PROVIDER: S-EPMC1218953 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Project description:We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.

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Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene.

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