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

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• 作者：Martin P. Vazquez ; David Mualem ; Natalia Bercovich ; Michael Zeev Stern ; Benson Nyambega ; Omer Barda ; Dan Masiga ; Sachin Kumar Gupta ; Shulamit Michaeli ; Mariano J. Levin
• 关键词：<i>Trypanosoma cruzii> ; <i>Trypanosoma bruceii> ; <i>Transi>-splicing ; U2 auxiliary factor
• 刊名：Molecular and Biochemical Parasitology
• 出版年：2009
• 出版时间：April 2009
• 年：2009
• 卷：164
• 期：2
• 页码：137-146
• 全文大小：1639 K

文摘

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.