Accepted_test

The Human Genome Project concluded over 20 years ago. However, the complete repertoire of coding open reading frames (ORFs) remains uncertain. Among these, the identification of small open reading frames, those consisting of less than 100 codons, poses a significant challenge. Ribosome profiling has made a revolution in our understanding of translated regions of different RNA molecules. Among the plethora of small ORFs, particular attention is drawn to those located in the 5’ untranslated regions of mRNAs, commonly known as upstream ORFs. Upstream ORFs can regulate translation in cis, encode small functional peptides (microproteins), be a source of antigenic peptides, or serve as the origin for new proteins and their domains during the evolutionary process. It is estimated that nearly half of mammalian mRNAs contain upstream ORFs. Nevertheless, their functions largely remain understudied. Therefore, we performed a functional genomics screening to identify essential upstream ORFs in human cell lines. The CRISPR-Cas9 knockout screening enabled us to identify upstream ORF that play important roles in essential cellular processes. The following analysis indicates that these selected upstream ORFs have the capability to modulate the translation of main ORFs or potentially encode functional microproteins.