The 3′ end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpΔ56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpΔ56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpΔ56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.
|Number of pages||12|
|Journal||Nucleic Acids Research|
|State||Published - May 2008|
Bibliographical noteFunding Information:
We thank Kate Zaytseva and Mingxia Zhang for technical assistance and Charles Query, Stefan Stamm and members of the Rymond lab for their helpful comments on this article. The pRS414-BBP, Acc, Nde/Acc, and SD5 plasmids were generously provided by Michael Rosbash. This work was supported by National Institutes of Health award GM42476 to BCR and infrastructure support in proteomics from the National Science Foundation award EPS-0132295. Funding to pay the Open Access publication charges for this article was provided by National Institutes of Health award GM42476 to BCR.
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