Seminar announcement 17th September 2014
CRISPR/Cas based tagging of a de novo evolved ORF, within the Reep6 gene in a mouse cell line
An important question in evolutionary biology is the origin of new genes. An increasing body of evidence suggests that de novo origination of a new gene from noncoding sequences may be much more frequent than previously thought. One of the mechanisms for de novo gene origination is a process called overprinting. It creates new proteins from a frameshifted open reading frame within a gene that already encodes a protein. In this study we present a strategy for verifying the translation of such putative de novo proteins. For this purpose we have used the genome-editing technology based on the CRISPR/Cas system, coupled with fusion-tag (FLAGTM-tag) technology.
Results of bioinformatic analysis showed the occurrence of an alternative reading frame within an existing Reep6 (receptor accessory protein) gene, suggestive of having acquired a new function at the boreoeutherian divergence. However, the protein product of the de novo originated reading frame has yet to be verified. The reading frame of the last exon of de novo ORF is 350 nt longer than the reading frame of the existing Reep6 gene ORF, i.e. the 3’ ends of last exon do not overlap. We have designed a CRISPR/Cas construct for site-directed homologous recombination to fuse the C-terminus of the de novo originated ORF to an eight amino acids long FLAGTM-tag and check for expression of this specific tag. This construct was used to transform a cell line. We find indeed evidence for translation of the FLAGTM-tag, implying that the new ORF is recognized in the cells used. This provides a proof of principle for the systematic identification of alternative protein products in overprinted genes.