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C. C. Query - One of the best experts on this subject based on the ideXlab platform.
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a novel u2 and u11 u12 snrnp Protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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A novel U2 and U11/U12 snRNP Protein that associates with the pre-mRNA branch site
EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2-and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immunoprecipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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Three recognition events at the branch-site adenine.
The EMBO Journal, 1996Co-Authors: C. C. Query, Scott A Strobel, Phillip A. SharpAbstract:An adenosine at the branch site, the nucleophile for the first transesterification step of splicing, is nearly invariant in mammalian pre-mRNA introns. The chemical groups on the adenine base were varied systematically and assayed for formation of early spliceosome complexes and execution of the first and second steps of splicing. Recognition of constituents of the adenine is critical in formation of a U2 snRNP-containing complex on a minimal branch-site oligonucleotide. Furthermore, the efficiencies of the first and second chemical steps have different dependencies on the functional groups of the adenine. In total, the chemical groups on the adenine base at the branch site are differentially recognized during at least three different processes in the splicing of pre-mRNA. Moreover, a Protein, P14, interacts with the adenine in a base-specific fashion and may mediate early recognition of this base.
Cindy L Will - One of the best experts on this subject based on the ideXlab platform.
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Molecular Architecture of the MultiProtein Splicing Factor SF3b
Science (New York N.Y.), 2003Co-Authors: Monika M. Golas, Cindy L Will, Reinhard Luhrmann, Bjoern Sander, Holger StarkAbstract:The splicing factor SF3b is a multiProtein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoProtein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of Protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the Protein P14 is located in the central cavity of the complex. The 22 tandem helical repeats of the Protein SF3b155 are located in the outer shell of the complex enclosing P14.
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a novel u2 and u11 u12 snrnp Protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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A novel U2 and U11/U12 snRNP Protein that associates with the pre-mRNA branch site
EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2-and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immunoprecipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.
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Improved identification of enriched peptide–RNA cross-links from ribonucleoProtein particles (RNPs) by mass spectrometry
Nucleic acids research, 2007Co-Authors: Eva Kühn-hölsken, Reinhard Luhrmann, Olexandr Dybkov, B. Sander, Henning UrlaubAbstract:Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized Protein–RNA contact sites in native ribonucleoProtein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide–RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the crosslinked species. We demonstrate the feasibility of this strategy by MS analysis of enriched peptide–RNA crosslinks from UV-irradiated reconstituted [15.5K-61KU4atacsnRNA] snRNPs and native U1snRNPs. Applying our approach to a partial complex of U2snRNP allowed us to identify the contact site between the U2snRNP-specific Protein P14/ SF3b14a and the branch-site interacting region (BSiR) of U2snRNA.
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Molecular Architecture of the MultiProtein Splicing Factor SF3b
Science (New York N.Y.), 2003Co-Authors: Monika M. Golas, Cindy L Will, Reinhard Luhrmann, Bjoern Sander, Holger StarkAbstract:The splicing factor SF3b is a multiProtein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoProtein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of Protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the Protein P14 is located in the central cavity of the complex. The 22 tandem helical repeats of the Protein SF3b155 are located in the outer shell of the complex enclosing P14.
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a novel u2 and u11 u12 snrnp Protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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A novel U2 and U11/U12 snRNP Protein that associates with the pre-mRNA branch site
EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2-and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immunoprecipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
Nikos F. Katopodis - One of the best experts on this subject based on the ideXlab platform.
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a novel u2 and u11 u12 snrnp Protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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A novel U2 and U11/U12 snRNP Protein that associates with the pre-mRNA branch site
EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2-and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immunoprecipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
Claudia Schneider - One of the best experts on this subject based on the ideXlab platform.
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a novel u2 and u11 u12 snrnp Protein that associates with the pre mrna branch site
The EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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A novel U2 and U11/U12 snRNP Protein that associates with the pre-mRNA branch site
EMBO Journal, 2001Co-Authors: Cindy L Will, Nikos F. Katopodis, Andrew M Macmillan, Gitte Neubauer, Reinhard Luhrmann, Claudia Schneider, Matthias Wilm, C. C. QueryAbstract:Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa Protein (P14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this Protein by microsequencing a 14 kDa Protein isolated from U2-type spliceosomes. This Protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded Protein precipitated the 14 kDa Protein cross-linked to the branch adenosine, confirming the identity of the P14 cDNA. A combination of immunoblotting, Protein microsequencing and immunoprecipitation revealed that P14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2-and U12-type pre-mRNAs, respectively. P14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immunoprecipitations indicated that P14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
