Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1

Early in the assembly of the spliceosome of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, w...

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Autores principales:	Vazquez, M.P., Mualem, D., Bercovich, N., Stern, M.Z., Nyambega, B., Barda, O., Masiga, D., Gupta, S.K., Michaeli, S., Levin, M.J.
Formato:	JOUR
Materias:	Trans-splicing Trypanosoma brucei Trypanosoma cruzi U2 auxiliary factor protein Sf1 protein u2af35 protein U2AF65 protozoal protein unclassified drug article cell fractionation controlled study molecular cloning nonhuman priority journal protein analysis protein expression protein function protein localization protein protein interaction RNA splicing Amino Acid Sequence Animals Cell Fractionation Cloning, Molecular Conserved Sequence Gene Expression Gene Silencing Models, Molecular Molecular Sequence Data Protein Interaction Mapping Protein Structure, Tertiary Protozoan Proteins RNA Splicing RNA-Binding Proteins Sequence Homology, Amino Acid Trypanosoma brucei brucei Two-Hybrid System Techniques Eukaryota Mammalia Trypanosomatidae
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_01666851_v164_n2_p137_Vazquez
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_01666851_v164_n2_p137_Vazquez
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spelling	todo:paper_01666851_v164_n2_p137_Vazquez2023-10-03T15:04:15Z Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1 Vazquez, M.P. Mualem, D. Bercovich, N. Stern, M.Z. Nyambega, B. Barda, O. Masiga, D. Gupta, S.K. Michaeli, S. Levin, M.J. Trans-splicing Trypanosoma brucei Trypanosoma cruzi U2 auxiliary factor protein Sf1 protein u2af35 protein U2AF65 protozoal protein unclassified drug article cell fractionation controlled study molecular cloning nonhuman priority journal protein analysis protein expression protein function protein localization protein protein interaction RNA splicing Trypanosoma brucei Trypanosoma cruzi Amino Acid Sequence Animals Cell Fractionation Cloning, Molecular Conserved Sequence Gene Expression Gene Silencing Models, Molecular Molecular Sequence Data Protein Interaction Mapping Protein Structure, Tertiary Protozoan Proteins RNA Splicing RNA-Binding Proteins Sequence Homology, Amino Acid Trypanosoma brucei brucei Trypanosoma cruzi Two-Hybrid System Techniques Eukaryota Mammalia Trypanosoma brucei Trypanosoma cruzi Trypanosomatidae Early in the assembly of the spliceosome of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 co-fractionated in a complex of approximately 400 kDa and U2AF65 was affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or even undetectable. © 2009 Elsevier B.V. All rights reserved. Fil:Vazquez, M.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Bercovich, N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_01666851_v164_n2_p137_Vazquez
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Trans-splicing Trypanosoma brucei Trypanosoma cruzi U2 auxiliary factor protein Sf1 protein u2af35 protein U2AF65 protozoal protein unclassified drug article cell fractionation controlled study molecular cloning nonhuman priority journal protein analysis protein expression protein function protein localization protein protein interaction RNA splicing Trypanosoma brucei Trypanosoma cruzi Amino Acid Sequence Animals Cell Fractionation Cloning, Molecular Conserved Sequence Gene Expression Gene Silencing Models, Molecular Molecular Sequence Data Protein Interaction Mapping Protein Structure, Tertiary Protozoan Proteins RNA Splicing RNA-Binding Proteins Sequence Homology, Amino Acid Trypanosoma brucei brucei Trypanosoma cruzi Two-Hybrid System Techniques Eukaryota Mammalia Trypanosoma brucei Trypanosoma cruzi Trypanosomatidae
spellingShingle	Trans-splicing Trypanosoma brucei Trypanosoma cruzi U2 auxiliary factor protein Sf1 protein u2af35 protein U2AF65 protozoal protein unclassified drug article cell fractionation controlled study molecular cloning nonhuman priority journal protein analysis protein expression protein function protein localization protein protein interaction RNA splicing Trypanosoma brucei Trypanosoma cruzi Amino Acid Sequence Animals Cell Fractionation Cloning, Molecular Conserved Sequence Gene Expression Gene Silencing Models, Molecular Molecular Sequence Data Protein Interaction Mapping Protein Structure, Tertiary Protozoan Proteins RNA Splicing RNA-Binding Proteins Sequence Homology, Amino Acid Trypanosoma brucei brucei Trypanosoma cruzi Two-Hybrid System Techniques Eukaryota Mammalia Trypanosoma brucei Trypanosoma cruzi Trypanosomatidae Vazquez, M.P. Mualem, D. Bercovich, N. Stern, M.Z. Nyambega, B. Barda, O. Masiga, D. Gupta, S.K. Michaeli, S. Levin, M.J. Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
topic_facet	Trans-splicing Trypanosoma brucei Trypanosoma cruzi U2 auxiliary factor protein Sf1 protein u2af35 protein U2AF65 protozoal protein unclassified drug article cell fractionation controlled study molecular cloning nonhuman priority journal protein analysis protein expression protein function protein localization protein protein interaction RNA splicing Trypanosoma brucei Trypanosoma cruzi Amino Acid Sequence Animals Cell Fractionation Cloning, Molecular Conserved Sequence Gene Expression Gene Silencing Models, Molecular Molecular Sequence Data Protein Interaction Mapping Protein Structure, Tertiary Protozoan Proteins RNA Splicing RNA-Binding Proteins Sequence Homology, Amino Acid Trypanosoma brucei brucei Trypanosoma cruzi Two-Hybrid System Techniques Eukaryota Mammalia Trypanosoma brucei Trypanosoma cruzi Trypanosomatidae
description	Early in the assembly of the spliceosome of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 co-fractionated in a complex of approximately 400 kDa and U2AF65 was affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or even undetectable. © 2009 Elsevier B.V. All rights reserved.
format	JOUR
author	Vazquez, M.P. Mualem, D. Bercovich, N. Stern, M.Z. Nyambega, B. Barda, O. Masiga, D. Gupta, S.K. Michaeli, S. Levin, M.J.
author_facet	Vazquez, M.P. Mualem, D. Bercovich, N. Stern, M.Z. Nyambega, B. Barda, O. Masiga, D. Gupta, S.K. Michaeli, S. Levin, M.J.
author_sort	Vazquez, M.P.
title	Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
title_short	Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
title_full	Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
title_fullStr	Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
title_full_unstemmed	Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1
title_sort	functional characterization and protein-protein interactions of trypanosome splicing factors u2af35, u2af65 and sf1
url	http://hdl.handle.net/20.500.12110/paper_01666851_v164_n2_p137_Vazquez
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Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1

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