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

Martin P. Vazquez; David Mualem; Natalia Bercovich; Michael Zeev Stern; Benson Nyambega; Omer Barda; Dan Masiga; Sachin Kumar Gupta; Shulamit Michaeli; Mariano J. Levin

doi:10.1016/j.molbiopara.2008.12.009

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

Martin P. Vazquez, David Mualem, Natalia Bercovich, Michael Zeev Stern, Benson Nyambega, Omer Barda, Dan Masiga, Sachin Kumar Gupta, Shulamit Michaeli, Mariano J. Levin

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Abstract

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.

Original language	English
Pages (from-to)	137-146
Number of pages	10
Journal	Molecular and Biochemical Parasitology
Volume	164
Issue number	2
DOIs	https://doi.org/10.1016/j.molbiopara.2008.12.009
State	Published - Apr 2009

Bibliographical note

Funding Information:
This work was mainly supported by grants from World Health Organization/Special Program for Research and Training in Tropical Diseases (WHO/TDR)- South South Initiative (SSI-WHO/TDR), a Mini-Grant of the Howard Hughes Medical Institute (Chevy Chase, MD, USA), the Chaire Internationale de Recherche Blaise Pascale (Region Ile de France, France), the SMMAD 40402 Project of the European Community, the FONCYT-PICT 01-06803 project to MJL, and FONCYT–PICT REDES 2003-00300, UBACyT X-153 (University of Buenos Aires) and PIP-CONICET 5492 to MV. MV and MJL are investigators of CONICET (Argentinean National Research Council). We acknowledge technical support from technicians Sonia Lafon and Maria de los Angeles Curto, both from CONICET. S.M is an HHMI International Scholar in Molecular Parasitology and this study was also supported by a grant from United States-Israel Binational Science foundation. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases. MJL has been International Research Scholar from the Howard Hughes Medical Institute, Chevy Chase, MD, USA (2002–2006) and Professor of a Blaise Pascal International Chair of Research, Fondation ENS-Region Ile de France, France (2005–2006). In addition, MJL was European Community Marie Curie International Fellow at Institut Cochin, Paris, France during the period September 2007–March 2008. Appendix A

Keywords

Trans-splicing
Trypanosoma brucei
Trypanosoma cruzi
U2 auxiliary factor

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10.1016/j.molbiopara.2008.12.009

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@article{4c4dfc1b4fb1412b9e9fa622f2998609,

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

abstract = "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.",

keywords = "Trans-splicing, Trypanosoma brucei, Trypanosoma cruzi, U2 auxiliary factor",

author = "Vazquez, {Martin P.} and David Mualem and Natalia Bercovich and Stern, {Michael Zeev} and Benson Nyambega and Omer Barda and Dan Masiga and Gupta, {Sachin Kumar} and Shulamit Michaeli and Levin, {Mariano J.}",

note = "Funding Information: This work was mainly supported by grants from World Health Organization/Special Program for Research and Training in Tropical Diseases (WHO/TDR)- South South Initiative (SSI-WHO/TDR), a Mini-Grant of the Howard Hughes Medical Institute (Chevy Chase, MD, USA), the Chaire Internationale de Recherche Blaise Pascale (Region Ile de France, France), the SMMAD 40402 Project of the European Community, the FONCYT-PICT 01-06803 project to MJL, and FONCYT–PICT REDES 2003-00300, UBACyT X-153 (University of Buenos Aires) and PIP-CONICET 5492 to MV. MV and MJL are investigators of CONICET (Argentinean National Research Council). We acknowledge technical support from technicians Sonia Lafon and Maria de los Angeles Curto, both from CONICET. S.M is an HHMI International Scholar in Molecular Parasitology and this study was also supported by a grant from United States-Israel Binational Science foundation. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases. MJL has been International Research Scholar from the Howard Hughes Medical Institute, Chevy Chase, MD, USA (2002–2006) and Professor of a Blaise Pascal International Chair of Research, Fondation ENS-Region Ile de France, France (2005–2006). In addition, MJL was European Community Marie Curie International Fellow at Institut Cochin, Paris, France during the period September 2007–March 2008. Appendix A ",

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AU - Mualem, David

AU - Bercovich, Natalia

AU - Stern, Michael Zeev

AU - Nyambega, Benson

AU - Barda, Omer

AU - Masiga, Dan

AU - Gupta, Sachin Kumar

AU - Michaeli, Shulamit

AU - Levin, Mariano J.

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PY - 2009/4

Y1 - 2009/4

N2 - 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.

AB - 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.

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KW - Trypanosoma brucei

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

Abstract

Bibliographical note

Keywords

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