The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3' UTR C-rich motifs.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a member of the hnRNP family and contains three canonical K-homology (KH) domains. hnRNP E1 acts as a cancer antagonist by regulating specific transcripts, including the canonical oncogenic driver p53, as well as p21. This study aims to elucidate the molecular mechanisms underlying hnRNP E1-mediated regulation of p53 and p21, which remain largely unexplored. Here, RNA immunoprecipitation and photoaffinity crosslinking assays revealed that hnRNP E1 directly binds p53 and p21 mRNAs via specific C-rich RNA motifs in their 3'-UTRs, enhancing RNA stability and translation through increased polyribosome loading, as confirmed by polyribosome fractionation assays. Domain-deletion-based genetic mapping indicated that hnRNP E1 modulates both p53 and p21 expression primarily via its KH1 and KH2 domains. Photoaffinity crosslinking experiments further confirmed that KH1 and KH2 domains independently interact with the 3'-UTRs of both p53 and p21 mRNA, mimicking the functional activity of full-length hnRNP E1 in in vitro translation and cell-based luciferase reporter assays. Functional assays revealed that KH1 and KH2 domains of hnRNP E1 modulate p53 and p21 via UTR-guided mechanisms, leading to reduced proliferation, colony formation, and increased apoptosis. Upregulation of apoptotic markers (Bax, pro-caspase-8, caspase-3) was observed as a regulatory resultant of KH1 and KH2 domain-guided p53/p21 interaction. By establishing p53 and p21 as active components of the hnRNP E1 transcriptome, this study identifies KH domains as promising therapeutic candidates, warranting future investigation.