| 1. |
- Estmer Nilsson, Camilla, et al.
(author)
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The adenovirus E4-ORF4 splicing enhancer protein interacts with a subsetof phosphorylated SR proteins
- 2001
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In: EMBO Journal. - 0261-4189 .- 1460-2075. ; 20, s. 864-871
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Journal article (peer-reviewed)abstract
- SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation
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| 2. |
- Estmer Nilsson, Camilla
(author)
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Viral Control of SR Protein Activity
- 2001
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Doctoral thesis (other academic/artistic)abstract
- Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing.Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.
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| 3. |
- Huang, Tien-Sheng, et al.
(author)
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Functional inactivation of the SR family of splicing factors during a vaccinia virus infection
- 2002
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In: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 3:11, s. 1088-1093
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Journal article (peer-reviewed)abstract
- SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.
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| 4. |
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| 5. |
- Kanopka, Arvydas, et al.
(author)
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Regulation of adenovirus alternative RNAsplicing by dephosphorylation of SR proteins
- 1998
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 393:6681, s. 185-187
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Journal article (peer-reviewed)abstract
- SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.
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| 6. |
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| 7. |
- Petersen-Mahrt, Svend K, et al.
(author)
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The splicing factor-associated protein, p32, regulates RNA splicing byinhibiting ASF/SF2 RNA binding and phosphorylation
- 1999
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In: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 18:4, s. 1014-1024
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Journal article (peer-reviewed)abstract
- The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.
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| 8. |
- Semenyuk, Andrey, et al.
(author)
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Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays
- 2006
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 356:1, s. 132-141
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Journal article (peer-reviewed)abstract
- A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.
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| 9. |
- Semenyuk, Andrey, et al.
(author)
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Synthesis of RNA Using 2'-O-DTM Protection
- 2006
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 128:38, s. 12356-12357
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Journal article (peer-reviewed)abstract
- tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2′-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2′-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5′-O-DMTr function can be used for high-throughput cartridge RNA purification.
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