Cytoplasmic polyadenylation in S. pombe
Cid1 is a cytoplasmic member of a novel class of regulatory poly(A) polymerases discovered recently in yeast, worms and vertebrates. Previous genetic studies in the fission yeast, Schizosaccharomyces pombe, suggested a role for Cid1 in the checkpoint response to replication stress, but it was not known how a poly(A) polymerase might contribute to this response. Further investigations into the mode of action of Cid1 were therefore undertaken in this study. Cid1 is likely to target specific RNAs for polyadenylation; potential RNA substrates were identified using the complementary methods of microarray hybridisation and whole proteome analysis using two-dimensional liquid chromatography. These experiments revealed that Cid1 does not affect RNAs during normal, unperturbed growth but instead alters the expression of specific subsets of genes during replication stress. Many RNAs affected by Cid1 in these circumstances were cell-cycle dependent and telomeric transcripts, including those encoding histones and a novel RecQ helicase, Rqh2. As Cid1 lacks an RNA recognition motif, it is unlikely to bind selectively to RNA targets on its own. Cid1-interacting proteins were identified using yeast two-hybrid and tandem affinity purification methods. From these studies, novel members of a Cid1 complex have been discovered including: a previously uncharacterised metallo-beta-lactamase, RNA-binding proteins, ribosomal proteins and a telomere-binding protein. Together, these approaches are leading to a model for the role of cytoplasmic polyadenylation by Cid1 in checkpoint control.