RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not

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.

Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

“…Recruitment of CNOT by RNA-binding factors has emerged as an important mechanism of posttranscriptional regulation (39,45,(77)(78)(79)(80). Utilization of CNOT by Pum orthologs has been reported in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster and mammals (11,43,44,72,(81)(82)(83), and thus represents an evolutionarily conserved mechanism. Like Drosophila Pum, the highly conserved RBD of Pum orthologs spanning from yeast to human universally interact with Pop2 orthologs.…”

Records of RNA localization through covalent tagging

“…These findings mirror analyses of RNA-protein interactions using tagging, in which the number of uridines added to an individual RNA molecule correlated well with its binding affinity for the protein, as determined in vitro. 26,29 Other variables, such as the structure of specific RNAs, may also effect tagging efficiency.…”

“…When the chimeric protein bound an RNA molecule in vivo, the end was "tagged" with uridine residues. The number of uridines added to each mRNA molecule mirrored the affinity of its sites for the RBP, and likely the integrated dwell time of the RBP on that particular RNA molecule 26 , as observed in vitro 29 .…”

The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

“…4D) could not accommodate a C without losing the double H-bond to the O4 Overall, it looks like DND1's tandem RRMs demonstrate a certain plasticity for RNA recognition where a range of sequences can be bound, but a Y3A4Y5U6 is necessary for high affinity binding. Such high affinity binding could be a prerequisite for the activation of downstream processes like the recruitment of regulatory factors (Webster et al 2019).Here, we propose that the tandem RRMs bind RNA in a two-step mechanism. In a first step a range of sequences may be preselected by low affinity binding in order to attach DND1 to scan the 3'UTR ( Fig.…”