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, USA.
RNA. 2024 Jun 17;30(7):866-890. doi: 10.1261/rna.079813.123.
The sequence-specific RNA-binding protein Pumilio (Pum) controls development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-β, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.
序列特异性 RNA 结合蛋白 Pumilio（Pum）控制着发育；然而，它调节的 mRNA 网络仍然没有被完全描述。在这项研究中，我们使用敲低和敲除方法，结合 RNA-seq 来测量 Pum 对培养细胞转录组的影响。我们还使用一种改进的 RNA 免疫共沉淀方法来鉴定胚胎中与 Pum 结合的 mRNAs。将这些数据集与跨转录组的 Pum 结合基序的位置进行整合，揭示了参与神经、肌肉、翅膀和生殖细胞发育以及细胞增殖的新的直接 Pum 靶基因。这些基因包括 Wnt、TGF-β、MAPK/ERK 和 Notch 信号通路、DNA 复制和脂质代谢的组成部分。我们鉴定了由 CCR4-NOT 脱腺苷酶复合物调节的 mRNAs，这是 Pum 介导的抑制的关键因素，并观察到 Pum：CCR4-NOT 靶 mRNAs 的一致调节。计算模型表明，Pum 结合、结合位点数量、聚类和序列背景是调节的重要决定因素。相比之下，我们表明，直接 mRNA 靶标的对 Pum 介导的抑制的响应不受最佳同义密码子含量的影响。此外，与流行的模型相反，我们没有检测到 CCR4-NOT 在降解低密码子优化的 mRNAs 中的作用。总之，这项工作的结果为 Pum 调控网络和机制以及影响 Pum 介导调控效率的参数提供了新的见解。
