eIF3 Engages with 3’-UTR Termini of Highly Translated mRNAs in Neural Progenitor Cells [APA-Seq]. eIF3 Engages with 3’-UTR Termini of Highly Translated mRNAs in Neural Progenitor Cells [APA-Seq]

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA)-Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3’-UTR termini in highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs. Overall design: To investigate the transcripts that eIF3 binds to in NPCs, we performed Quick-irCLIP in undifferentiated and early differentiated NPCs. We then performed alternative polyadenylation sequencing (APA-Seq) to validate our findings of eIF3 crosslinking at 3'-UTRs. Finally, we performed Ribosome profiling to determine that eIF3 crosslinking at 3'-UTRs in NPCs is correlated with high translation activity. Grantee: Santi Mestre-Fos Grant ID: EDUC4-12790 Funding Source: California Institute for Regenerative Medicine

干细胞分化（Stem cell differentiation）过程中，蛋白质合成会发生全局性上调，以满足特化细胞类型的功能需求。然而，这种翻译爆发的分子机制，以及翻译起始因子（initiation factors）在其中的参与方式，目前仍未完全阐明。本研究旨在探究真核翻译起始因子3（eukaryotic initiation factor 3, eIF3）在人类多能干细胞（human pluripotent stem cell, hPSC）诱导的神经前体细胞（neural progenitor cells, NPCs）早期分化中的功能。
我们采用Quick-irCLIP与可变多聚腺苷酸化测序（alternative polyadenylation, APA-Seq）技术，证实eIF3可与神经前体细胞内多种神经相关mRNA发生交联。研究数据显示，eIF3主要结合多种mRNA异构体的3'非翻译区（3' untranslated region, 3'-UTR）末端区域，该区域紧邻poly(A)尾。通过核糖体谱（ribosome profiling）分析可知，mRNA的3'-UTR末端区域的eIF3交联水平较高，与翻译活性上调显著相关。我们以转录调控因子DNA结合抑制因子2（inhibitor of DNA binding 2, ID2）的mRNA为例，发现在神经前体细胞早期分化过程中，该mRNA的翻译活性与eIF3交联水平均显著升高。此外，我们发现eIF3在3'-UTR末端的结合依赖于多聚腺苷酸化过程。本研究结果表明，在高翻译活性的mRNA中，eIF3可结合其3'-UTR末端区域，这为mRNA环化参与神经前体细胞mRNA翻译调控的机制提供了实验支持。
整体实验设计：为探究神经前体细胞内eIF3的结合转录本，我们分别在未分化及早期分化的神经前体细胞中开展Quick-irCLIP实验。随后通过可变多聚腺苷酸化测序（APA-Seq）验证eIF3在3'-UTR区域的交联现象。最后利用核糖体谱分析，证实神经前体细胞中eIF3在3'-UTR区域的交联水平与翻译活性呈正相关。
资助信息：项目负责人：桑蒂·梅斯特雷-福斯（Santi Mestre-Fos）；项目编号：EDUC4-12790；资助方：加利福尼亚再生医学研究所（California Institute for Regenerative Medicine）