Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

Gil, Anna; Sharp, Phillip A.; Jamison, Sharon F.; García-Blanco, Mariano A.

doi:10.1101/gad.5.7.1224

Cited by 251 publications

(239 citation statements)

References 72 publications

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“…Our finding of PTB in HFFs, G 0 HFFs, hTERT-HFFs and hTERT-RPEs was consistent with its widespread expression in multiple cell types (Gil et al, 1991). Intriguingly, the induction of PTB was preceded by the appearance of smaller PTB isoforms, which reacted with a monoclonal antibody against the PTB carboxy terminus.…”

Section: Discussionsupporting

confidence: 63%

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection

Adair

Liebisch

et al. 2004

Journal of General Virology

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Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G 0 ) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA. INTRODUCTIONThe human cytomegalovirus (HCMV) UL37 immediateearly (IE) gene locus encodes several protein isoforms, of which at least one, the UL37 exon 1 protein (pUL37x1), is essential for HCMV growth in humans (Hayajneh et al., 2001a) and in cultured human foreskin fibroblasts (HFFs) (Dunn et al., 2003;Yu et al., 2003). pUL37x1 shares aminoterminal sequences, including its anti-apoptotic domains, with the UL37 glycoprotein, gpUL37, and the UL37 medium protein, pUL37 M (Kouzarides et al., 1988;Goldmacher et al., 1999). While the carboxy terminus of gpUL37 is nonessential for HCMV growth in HFFs (Borst et al., 1999), it appears to play a role in HCMV growth in humans, as it does for mouse CMV pathogenesis in vivo (Lee et al., 2000;Hayajneh et al., 2001b). pUL37x1 is the product of the UL37x1 unspliced RNA and is the predominant UL37 protein produced throughout permissive HCMV infection of HFFs (Mavinakere & Colberg-Poley, 2004a). Nonetheless, gpUL37, the product of the UL37 3?4 kb spliced RNA is also produced, at lower abundance, and is N-glycosylated (Al- Barazi & ColbergPoley, 1996). Both HCMV UL37 proteins traffic dually into the endoplasmic reticulum (ER) and into mitochondria, where they block the release of cyt...

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Section: Discussionsupporting

confidence: 63%

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection

Adair

Liebisch

et al. 2004

Journal of General Virology

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“…The fact that PTB may be just one component of a larger complex is indicated by our finding that complex I is at least 150-kDa in size (K. Singh and L. R. Covey, unpublished data) and the presence of a different, but related complex that binds the E5Ј-HaeIII transcript. PTB is expressed ubiquitously and as three alternatively spliced isoforms in many different cells and tissue types (46,48). We have previously shown that complex I only forms in T cells after extended activation.…”

Section: Discussionmentioning

confidence: 99%

A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

Kosinski

Laughlin

Singh

et al. 2003

The Journal of Immunology

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CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of “regulated instability” of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3′ untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Δ1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Δ1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.

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“…Several isoforms and paralogs of PTB have been reported [10,40,41]. Brain-specific PTB is known to be a different gene product of PTB, i.e., a paralog of PTB.…”

Section: Ptb In Drm Fraction Of Replicon Cells Consists Of Multiple Imentioning

confidence: 99%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

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Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

Cited by 251 publications

References 72 publications

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection

A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

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