Nucleic Acids Research, 2003, Vol. 31, No. 6 1744-1752
© 2003 Oxford University Press
Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor
1 Department of Molecular Biology and Microbiology and 2 Department of Biochemistry, Tufts University School of Medicine and Sackler Graduate School of Biomedical Sciences, Boston, MA 02111, USA
*To whom correspondence should be addressed. Tel: +1 617 6366935; Fax: +1 617 636 0337; Email: claire.moore{at}tufts.edu
Present address:
Steffen Helmling, NOXXON Pharma AG, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany
Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers. Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3' end processing. In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro. We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation. Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro. In vitro assays showed that the N-terminus is necessary for both processing steps. Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA–Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1. Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.
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