Evidence for Widespread Presence of Hidden yet Functional Novel and Non-canonical Human Transcripts

Fraction of functional sequence in human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, like transcription, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse-genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. Altogether, these results argue that many unannotated human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions. Overall design: Used a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool for identification of novel functional genomic elements