POST-TRANSCRIPTIONAL REGULATION OF KRAS GENE: MECHANISM INVOLVING 5'UTR RNA G-QUADRUPLEXES, LONG NONCODING RNA AND NUCLEAR RIBONUCLEOPROTEIN A1

Previous studies have shown that G-quadruplex (G4) DNA plays a crucial role in the regulation of gene expression. Here we show that the expression of the human KRAS oncogene is not only controlled by promoter G4 DNA, which serves as a platform for transcription factor recruitment, but also by G4 RNA (RG4) structures within the 5'UTR of messanger RNA, which control mRNA stability. The presence of these folded structures in vivo was demonstrated by ChIP-seq and RIP-seq experiments. Genetic abrogation of RG4 structures using CRISPR/Cas9 leads to increased mRNA levels, and point mutations that destabilise RG4s also lead to an increase in transcript levels. Bioinformatic analysis shows that the noncoding RNA HSALNT12722 contains a segment that is fully complementary to the 5'-UTR carrying the RG4 structures. The resulting RNA duplex is recognised and cleaved by RNase III. Furthermore, we could show that hybridisation of HSALNT12722 with the 5'UTR is mediated by hnRNPA1, which binds to the RG4 structures and unfolds them. Overall, our work demonstrates that the human KRAS oncogene is regulated both at the transcriptional level by G4 DNA and at the post-transcriptional level by RG4 structures and noncoding RNA, which determine the stability of KRAS mRNA. Overall design: Panc-1 cells treated or not for 16h with PDS (1µM). After incubation, cells were harvested and subjected to rG4-IP