Investigating the biological role of a protein is typically achieved with loss- or gain-of-function screens, or through comprehensive open reading frame (ORF) libraries that permit a systematic analysis of the entire protein-coding genome. However, these methods have limitations, such as a lack of functional insights and a bias towards shorter ORFs. Perutz PI Stefan Ameres explains: “Utilizing ORFeome libraries is inherently costly and challenging to generate and maintain due to the complexity involved, with at least 20,000 genes.” To address these challenges, Stefan, together with Ulrich Elling (IMBA), conceptualized a time and resource efficient approach inspired by gene trap methods, simplifying the systematic investigation of protein functionality.
“In ORFtag, we use a retroviral vector that integrates randomly in the genome. It contains a strong promoter, a resistance cassette, and a customizable tag followed by a splice donor site”, Stefan elaborates. “After integration, the promoter drives expression, and the splice donor site triggers splicing of the tag to a downstream exon encoded in a nearby gene.” This method allows researchers to generate fusion mRNAs of any target gene. He adds: “ORFtag is simple, cheap, and relatively fast. All you need are three vectors to target the three potential reading frames of the fused exon.”
To demonstrate the utility of ORFtag, Moritz Himmelsbach, PhD student in the Ameres lab and first author, took the project successfully from concept to bench, enabling him to identify many proteins with post-transcriptional gene regulatory activity. In collaboration with the labs of Alexander Stark (IMP) and Julius Brennecke (IMBA), the researchers adapted ORFtag for different applications, such as the identification of transcriptional activators and repressors, thereby enhancing its overall utility. Using ORFtag, the team discovered a previously unknown transcriptional activator, Zfp574 and found that oncogenic fusion proteins may often function as transcriptional transactivators.
Currently, the proof-of-concept studies have been performed in mouse embryonic stem cells, but the technology can in principle address any other cultured mammalian cells. However, one limitation of ORFtag is that it can only target genes with introns. Stefan notes: “Approximately 6 % of genes cannot be addressed because they do not undergo splicing after transcription. However, most of these genes are confined in their function and encode for histones and sensory receptors, leaving more than 90% of the protein-coding genes as potentially taggable by ORFtag.” Hence, ORFtag represents a cost-effective and time-saving method for systematically and comprehensively investigating protein function.
DOI: 10.1038/s41592-024-02339-x