Haugen Rebecca J, Barnier Catherine, Elrod Nathan D, Luo Hua, Jensen Madeline K, Ji Ping, Smibert Craig A, Lipshitz Howard D, Wagner Eric J, Lydia Freddolino P, Goldstrohm Aaron C
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, 48109.
bioRxiv. 2023 Aug 30:2023.08.29.555372. doi: 10.1101/2023.08.29.555372.
The sequence-specific RNA-binding protein Pumilio controls development of ; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.
序列特异性RNA结合蛋白Pumilio控制着 的发育；然而，其调控的mRNA网络仍未完全明确。在本研究中，我们利用基因敲低和基因敲除方法结合RNA测序来测量Pumilio对 细胞转录组的影响。我们还使用了一种改进的RNA共免疫沉淀方法来鉴定 胚胎中与Pumilio结合的mRNA。将这些数据集与转录组中Pumilio结合基序的内容整合，揭示了涉及神经、肌肉、翅膀和生殖细胞发育以及细胞增殖的新的直接Pumilio靶基因。这些基因包括Wnt、TGF-β、MAPK/ERK和Notch信号通路的组成部分、DNA复制和脂质代谢。此外，我们鉴定了由CCR4-NOT去腺苷酸化酶复合体调控的mRNA，这是Pumilio介导的抑制作用中的一个关键因子，并观察到Pumilio:CCR4-NOT靶mRNA的协同调控。计算模型表明，Pumilio结合、结合位点数量、密度和序列背景是调控的重要决定因素。此外，靶mRNA中最优同义密码子的含量与Pumilio和CCR4-NOT调控呈现出显著的功能关系，表明mRNA固有的翻译效率和稳定性调节了它们对这些反式作用调控因子的反应。总之，这项工作的结果为Pumilio调控网络和机制以及影响Pumilio介导调控效率的参数提供了新的见解。
