ABSTRACT
Alternative Lengthening of Telomeres (ALT) utilizes a recombination mechanism and break-induced DNA synthesis to maintain telomere length without telomerase, but it is unclear how cells initiate ALT. TERRA, telomeric repeat-containing RNA, forms RNA:DNA hybrids (R-loops) at ALT telomeres. We show that depleting TERRA using an RNA-targeting Cas9 system reduces ALT-associated PML bodies, telomere clustering, and telomere lengthening. TERRA interactome reveals that TERRA interacts with an extensive subset of DNA repair proteins in ALT cells. One of TERRA interacting proteins, the endonuclease XPF, is highly enriched at ALT telomeres and recruited by telomeric R-loops to induce DNA damage response (DDR) independent of CSB and SLX4, and thus triggers break-induced telomere synthesis and lengthening. The attraction of BRCA1 and RAD51 at telomeres requires XPF in FANCM-deficient cells that accumulate telomeric R-loops. Our results suggest that telomeric R-loops activate DDR via XPF to promote homologous recombination and telomere replication to drive ALT.
Subject(s)
Telomerase , DNA , Endonucleases/metabolism , RNA , Telomerase/genetics , Telomerase/metabolism , Telomere/genetics , Telomere/metabolism , Telomere HomeostasisABSTRACT
Recent studies have suggested that DDX3 functions in antiviral innate immunity, but the underlying mechanism remains elusive. We previously identified target mRNAs whose translation is controlled by DDX3. Pathway enrichment analysis of these targets indicated that DDX3 is involved in various infections and inflammation. Using immunoblotting, we confirmed that PACT, STAT1, GNB2, Rac1, TAK1, and p38 mitogen-activated protein kinase (MAPK) proteins are downregulated by DDX3 knockdown in human monocytic THP-1 cells and epithelial HeLa cells. Polysome profiling revealed that DDX3 knockdown reduces the translational efficiency of target mRNAs. We further demonstrated DDX3-mediated translational control of target mRNAs by luciferase reporter assays. To examine the effects of DDX3 knockdown on macrophage migration and phagocytosis, we performed in vitro cell migration assay and flow cytometry analysis of the uptake of green fluorescent protein-expressing Escherichia coli in THP-1 cells. The DDX3 knockdown cells exhibited impaired macrophage migration and phagocytosis. Moreover, we used a human cytokine antibody array to identify the cytokines affected by DDX3 knockdown. Several chemokines were decreased considerably in DDX3 knockdown THP-1 cells after lipopolysaccharide or poly(I·C) stimulation. Lastly, we demonstrated that DDX3 is crucial for the recruitment of phagocytes to the site of inflammation in transgenic zebrafish.
