The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) interact with Ccr4-Not via multiple regions within low-complexity sequences, suggestive of a multipartite interface that extends beyond previously defined interactions. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. Analysis of binding kinetics reveals that this is primarily due to differences in dissociation rate constants. Consequently, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo and can be used for the prediction of mRNA targets.
All data generated during this study are included in the manuscript and supporting files. Source data are provided in Supplementary Files 1 and 2.
Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in YeastPLOS, 10.1371/journal.pbio.0020079.
Protein-RNA networks revealed through covalent RNA marks.Nature, nmeth.3651.
- Lori A Passmore
- Lori A Passmore
- Lori A Passmore
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Rachel Green, Johns Hopkins School of Medicine, United States
© 2019, Webster et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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