Unbiased screen of cap-independent translation initiation elements reveals new complexity of translational regulation

Although most eukaryotic mRNAs require the 5'-cap for translation, some mRNAs can also be translated through a cap-independent pathway. Here we developed a cell-based system to unbiasedly screen human transcriptome for sequences that drive cap-independent circRNA translation, and identified >10,000 endogenous sequences. These sites were found in all mRNA regions, with features distinct from canonical IRESs, and thus were defined as Cap-independent Translation Initiation sites (CiTI). The CiTIs at 5'-UTR tend to pair with 18S rRNA to promote translation. However, the CiTIs at 3'-UTR may function either as IRESs to drive downstream ORF translation, or as translation enhancers to promote upstream main ORF translation by unwinding the highly structured 5'-UTR. We further identified trans-acting factors that specifically bind CiTIs at 3'-UTR to promote upstream ORF translation, including several components of translation initiation complexes such as DHX29. Knockdown of DHX29 reduced translation of a specific set of mRNAs, including HIF1A whose translation is enhanced by a 3'-CiTI during hypoxia. Consistently, deletion of this 3'-CiTI from HIF1A can suppress neuroblastoma cell proliferation in vitro and in vivo. Collectively, the identification and study of CiTI revealed new diversity in translational regulation that may play key roles in mRNA translation during stress response.