Abstract
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.
Highlights
Shortening or removal of mRNA poly(A) tails represses gene expression in eukaryotes
We previously reported that Caf1 and Ccr4 have similar activities within the purified Ccr4-Not complex (Stowell et al, 2016)
To assess the limitations of predicting RNA-binding protein targets directly from mRNA sequence motif we examined ScPuf3 binding and the sequence features of transcripts that had motif 1, 2 or 3 in the 3’-UTR but encoded proteins not related to mitochondrial biogenesis (33 in total), as well as mRNAs related to mitochondrial biogenesis but with motif 4 in the 3’-UTR (33 transcripts)
Summary
Shortening or removal of mRNA poly(A) tails (deadenylation) represses gene expression in eukaryotes. RNA-binding proteins such as Pumilio/Puf and Tristetraprolin/TTP can recognize sequence motifs in mRNAs, and bind Ccr4-Not to target those RNAs for deadenylation (Wahle and Winkler, 2013). The set of transcripts targeted for repression and decay by a given RNA-binding protein is defined by the distribution of its binding motifs across the transcriptome, and often includes groups of mRNAs that encode functionally related proteins This generates RNA regulatory networks, structured such that a single protein can regulate the expression of numerous genes (Joshi et al, 2011; Keene, 2007; Lapointe et al, 2017; Wilinski et al, 2015). The nine-nucleotide ARE motif (UUAUUUAUU) was identified in unstable mRNAs encoding cytokines and lymphokines in human cells (Caput et al, 1986; Chen and Shyu, 1995) This sequence is recognized by tandem zinc finger (TZF) proteins including tristetraprolin (TTP). Our findings show that a substantially improved understanding of RNA-binding protein regulatory networks can be obtained through detailed analysis of motif quality
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