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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

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

Michael W Webster1, James Aw Stowell1, Lori A Passmore1

  • 1MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Elife
|January 3, 2019
PubMed

Insights

Researchers identified how specific RNA-binding proteins, Zfs1 and Puf3, interact with the Ccr4-Not complex to control mRNA stability. This interaction mechanism helps predict mRNA targets and understand gene regulation.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • The Ccr4-Not complex is crucial for regulating eukaryotic mRNA stability and translation by shortening poly(A) tails.
  • RNA-binding proteins (RBPs) confer specificity to Ccr4-Not activity by interacting with both the complex and target mRNAs, though the precise mechanisms remain unclear.

Purpose of the Study:

  • To investigate the molecular mechanisms by which RBPs, specifically Zfs1 and Puf3, interact with the Ccr4-Not complex to mediate selective mRNA deadenylation.
  • To explore how these interactions influence mRNA stability and to determine if motif quality can predict mRNA targets.

Main Methods:

  • Reconstitution of accelerated and selective deadenylation assays for RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs).
  • Investigation of interactions between fission yeast homologs Zfs1 (Tristetraprolin/TTP) and Puf3 (Pumilio/Puf) with the Ccr4-Not complex using low-complexity sequences.
  • Employment of a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs.
  • Analysis of binding kinetics, focusing on dissociation rate constants.

Main Results:

  • Zfs1 and Puf3 interact with Ccr4-Not through multiple regions within low-complexity sequences, forming a multipartite interface.
  • Puf3 demonstrates the ability to distinguish between RNAs with highly similar sequences, primarily due to differences in dissociation rates.
  • Motif quality significantly impacts mRNA stability for Puf3 targets in vivo.

Conclusions:

  • The study elucidates a novel, multipartite interaction mechanism between RBPs (Zfs1, Puf3) and the Ccr4-Not complex for targeted mRNA deadenylation.
  • Dissociation kinetics and motif quality are key determinants of Puf3-mediated mRNA decay.
  • These findings provide a basis for predicting mRNA targets based on motif characteristics and understanding post-transcriptional gene regulation.
Keywords:
S. cerevisiaeS. pombebiochemistrychemical biologydeadenylationgene expressionpoly(A) tail

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