A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs.
A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs.
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DOI：
10.1016/j.jbc.2022.102270
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发表时间：
2022-09
影响因子：
4.8
通讯作者：
Goldstrohm, Aaron C.
中科院分区：
文献类型：
--
作者：
Haugen, Rebecca J.;Arvola, Rene M.;Connacher, Robert P.;Roden, Richard T.;Goldstrohm, Aaron C.
Pumilio是一种序列特异性rna结合蛋白，控制果蝇的发育、干细胞命运和神经功能。Pumilio通过依赖于CCR4-NOT deadenylase复合物的方式破坏靶mrna的稳定，从而抑制蛋白表达。在之前的研究中发现，Pumilio的n端区域有三个独特的抑制域可以招募CCR4-NOT，但它们是如何招募CCR4-NOT的还不清楚。在这项研究中，我们确定了Pumilio第三个抑制域（称为RD3）活性所必需和充分的基序，该基序存在于所有亚型中，并具有保守的调节功能。我们确定了RD3的多个保守区域，这些区域在基于细胞的报告基因检测中对抑制活性很重要。通过酵母双杂交实验，我们发现RD3与CCR4-NOT复合物的Not1、Not2和Not3亚基的特定区域有接触。我们的研究结果表明，RD3通过保守的短线性相互作用基序介导与CCR4-NOT进行多价相互作用。具体来说，RD3中的两个苯丙氨酸残基与Not1建立了至关重要的接触，这对Not1的抑制活性至关重要。通过报告基因检测，我们还发现了三个被Pumilio抑制的新靶mrna，并表明RD3参与了它们的调控。总之，这些结果为Pumilio招募CCR4-NOT调节靶mrna表达的机制提供了重要的见解。
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.
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