A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:298 |
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| Enthalten in: |
The Journal of biological chemistry - 298(2022), 9 vom: 15. Sept., Seite 102270 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Haugen, Rebecca J [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 29.09.2022 Date Revised 18.10.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2022.102270 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM343704927 |
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| 100 | 1 | |a Haugen, Rebecca J |e verfasserin |4 aut | |
| 245 | 1 | 2 | |a A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs |
| 264 | 1 | |c 2022 | |
| 336 | |a Text |b txt |2 rdacontent | ||
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| 500 | |a Date Completed 29.09.2022 | ||
| 500 | |a Date Revised 18.10.2022 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a CCR4–NOT | |
| 650 | 4 | |a Pumilio | |
| 650 | 4 | |a deadenylase | |
| 650 | 4 | |a mRNA regulation | |
| 650 | 7 | |a Drosophila Proteins |2 NLM | |
| 650 | 7 | |a NOT1 protein, Drosophila |2 NLM | |
| 650 | 7 | |a Protein Isoforms |2 NLM | |
| 650 | 7 | |a RNA, Messenger |2 NLM | |
| 650 | 7 | |a RNA-Binding Proteins |2 NLM | |
| 650 | 7 | |a pum protein, Drosophila |2 NLM | |
| 650 | 7 | |a Phenylalanine |2 NLM | |
| 650 | 7 | |a 47E5O17Y3R |2 NLM | |
| 650 | 7 | |a CCR4 protein, Drosophila |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 650 | 7 | |a Ribonucleases |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 700 | 1 | |a Arvola, René M |e verfasserin |4 aut | |
| 700 | 1 | |a Connacher, Robert P |e verfasserin |4 aut | |
| 700 | 1 | |a Roden, Richard T |e verfasserin |4 aut | |
| 700 | 1 | |a Goldstrohm, Aaron C |e verfasserin |4 aut | |
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| 856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.jbc.2022.102270 |3 Volltext |
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