Ester Cannizzaro

ROLE OF DDX RNA HELICASES IN CANCER CELLS

Abstract

DEAD-BOX (DDX) RNA helicases are a large family of proteins characterized by the presence of a DEAD/H (Asp-Glu-Ala-Asp/His) motif. Their main function is to unwind double stranded RNA to promote downstream molecular events. They are involved in virtually all steps of RNA metabolism such as transcription, translation, RNA export and degradation, ribosome biogenesis and pre-mRNA splicing. The aim of my work was to investigate the functions of human DDX3X and DDX54 RNA helicases, particularly in in vitro cancer models.

To provide insight into their molecular functions, I identified RNAs bound by DDX3X and DDX54 in breast cancer (MCF7) cells by performing iCLIP experiments. This generated two very distinct RNA binding profiles: DDX3X preferentially bound exonic regions of mRNAs encoding translational factors, whilst DDX54 preferentially bound non-coding RNAs and intronic regions of mRNAs encoding nuclear proteins. Further bioinformatic analysis identified a few discrete binding motifs within DDX3X target RNAs. One of these, within the human JUND transcript, was validated as a DDX3X binding site using electrophoretic mobility shift assays.

Notably, the levels of proteins encoded by mRNAs bound by DDX3X were altered following knockdown of the helicase. These data highlight the importance of DDX3X in maintaining appropriate levels of certain proteins, which in turn may explain at least some of the changes in phenotype observed upon DDX3X knockdown. In this regard, I showed that knocking down DDX3X or DDX54 in MCF7 cells slowed cell proliferation by inducing a G1/S phase arrest. Furthermore, a CRISPR/Cas9 dropout screen in a leukaemia cell line (MLL-AF9) showed that both helicases are essential for growth of these cells. However, loss of DDX3X or DDX54 had little effect on proliferation of immortalized NIH3T3 cells indicating that their loss is not generally lethal. In subsequent CRISPR/Cas9 dropout screens in various other cancer cell lines, including some derived from solid tumours, DDX54 was found to be essential for growth of all cell lines. In contrast, DDX3X was required for proliferation of only a subset of these. Focusing on DDX3X, I identified that the integrity of the helicase's RNA binding domains is essential for growth and cell cycle progression of an acute myeloid leukaemia cell line (OCI-AML3). Overall, my findings shed mechanistic insight upon the role of DDX RNA helicases in cancer and identify DDX3X and DDX54 as potential targets for therapeutic intervention in certain cancers.