eIF3 Engages with 3’-UTR Termini of Highly Translated mRNAs in Neural Progenitor Cells [Quick-irCLIP]

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. 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