A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4–NOT deadenylase complex subunits to repress target mRNAs

Rebecca J. Haugen, René M. Arvola, Robert P. Connacher, Richard T. Roden, Aaron C. Goldstrohm

Research output: Contribution to journalArticlepeer-review

Abstract

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4–NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4–NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4–NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4–NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4–NOT to regulate the expression of target mRNAs.

Original languageEnglish (US)
Article number102270
JournalJournal of Biological Chemistry
Volume298
Issue number9
DOIs
StatePublished - Sep 2022

Bibliographical note

Funding Information:
This research was funded by the National Institute of General Medical Sciences, National Institutes of Health (grant no.: R01 GM105707; to A. C. G.). R. M. A. was supported by a fellowship (grant no.: DGE 1256260 ) from the National Science Foundation . Further support was provided by the University of Minnesota Medical School . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2022 The Authors

Keywords

  • CCR4–NOT
  • Pumilio
  • deadenylase
  • mRNA regulation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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