Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor.
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.
Tacahashi
Yoko
Y
Department of Molecular Biology and Microbiology, Tufts University School of Medicine and Sackler Graduate School of Biomedical Sciences, Boston, MA 02111, USA.
Helmling
Steffen
S
Moore
Claire L
CL
D001665:Binding Sites / Q000235:genetics; D005816:Genetic Complementation Test / Q000379:methods; D009154:Mutation; D010641:Phenotype; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics* / Q000378:metabolism
C450579:FIP1 protein, S cerevisiae; D012322:RNA Precursors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C450580:YTH1 protein, S cerevisiae; D039221:mRNA Cleavage and Polyadenylation Factors; D011061:Poly A