Regulation of innate immunity by DEAD box RNA helicase DDX3X

The innate immune system evolved to provide immediate protection from infection. Its operation involves the recognition of characteristic pathogen-associated molecular patterns (PAMPs) using a limited number of extracellular, membrane-associated or cytoplasmic receptors. Recognition is transmitted through a complex series of molecular interactions and enzymatic modifications to result in a pathogen-adapted innate immune response. In this process de novo synthesis of gene products plays an essential role. Many of the genes transcribed after microbe recognition encode cytokines, polypeptide mediators of the immune response. Among these are type I interferons (IFN-I). IFN-I direct innate antiviral immunity and contribute to the regulation of protective responses to nonviral pathogens. Synthesis and secretion of IFN-I is considered a hallmark of innate immune responses. In previous work we investigated signal transduction by pattern recognition receptors that results in activation of the IFN-beta gene, a member of the IFN-I gene family. We focused our attention on interactors of protein kinase TBK1, an essential component of the signaling pathway connecting innate antigen receptors with the IFN-beta gene. Our studies resulted in the identification DDX3X, a DEAD box RNA helicase. We were able to show that DDX3X is a TBK1 substrate, that it enhances IFN-beta gene expression and in doing so interacts with the IFN-beta promoter. These results identified DDX3X as novel component of the innate immune system. At the same time they revealed that the immunological function of DDX3X is independent of its helicase activity. The mechanism of action of DDX3X is therefore entirely unclear and, because without precedence, unknown territory. Our proposal describes experiments designed to clarify the mechanism by which DDX3X impacts on IFN-beta expression and on the innate immune response in general. They will also provide important information about the extent to which DDX3X reinforces antimicrobial defense. To address the former question we will identify nuclear interactors that will lead the way to DDX3X mode of operation. Specifically these interactors will reveal the regulatory impact of DDX3X on transcriptional activation of the IFN-beta gene. To reveal the impact of DDX3X on host defence we will stimulate cells and mice expressing no or mutant DDX3X with PAMPs or infect them with pathogens. This approach will reveal the significance of DDX3X for innate immunity. In conclusion our project will functionally characterize a new player in the immune system and fathom the potential of directed guidance of host defense through inhibition or enhancement of DDX3X activity.

Our work established transcriptional regulation by the RNA helicase DDX3X as an important component of antimicrobial immunity and identified an essential role of the helicase in the generation of the cells constituting the immune system. The innate immune system evolved to provide immediate protection from infection. It recognizes characteristic conserved features of microbes and responds to recognition with an antimicrobial response that directs signals to the cell nucleus to cause de novo synthesis of gene products. Among these are type I interferons (IFN-I). IFN-I direct innate antiviral immunity and contribute to the regulation of protective responses to nonviral pathogens. In previous work we identified the DExD/H box RNA helicase, DDX3X as a modulator of IFN-I gene transcription in cells infected with the intracellular bacterial pathogen Listeria monocytogenes. Members of the DExD/H box helicases perform a wide variety of functions in RNA metabolism. Furthermore, an increasing number of family members are recognized as components of the innate immune system involved in nucleic acid sensing, signal transduction and transcriptional regulation. Enhancement of IFN-I transcription occurred even after elimination of the DDX3X helicase activity by mutation, suggesting a non-canonical mode of action. DDX3 occurs as two isoforms, the X-chromosome encoded DDX3X and the Y-chromosome-encoded DDX3Y. We have shown that the two isoforms are partially redundant, both regarding essential functions for cell survival and in the innate immune system. In addition to IFN-I, the DDX3 isoforms regulate a range of antimicrobial genes and deletion of DDX3X in hematopoietic cells of mice results in strongly decreased innate immunity against the bacterial pathogen Listeria monocytogenes (Lm), but has little impact on resistance to vesicular stomatitis virus (VSV). Analysis of hematopoiesis in mice lacking hematopoietic DDX3X showed that generation of cells belonging to the lymphoid lineage is strongly impaired and this is particularly true for natural killer (NK) cells. Consistent with this defect, such mice produced strongly reduced amounts of interferon gamma (IFN?) during infection with L. monocytogenes. Early NK-derived IFN? is considered an important protective mechanism of the innate immune system. To examine how DDX3X stimulates transcription we performed a mass-spectrometry-based search for interacting proteins. In addition to proteins reflecting the typical DDX3X helicase function, this search produced interactors involved in gene regulation. In particular, proteins involved in determining chromatin structure and the RNA polymerase transcription cycle were found. Based on these observations we speculate that DDX3X modulates gene activity by affecting the presence or position of nucleosomes and, most likely connected with this, the processes of transcriptional initiation and elongation by RNA polymerase II.