Abstract
RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells.
Highlights
RNA:DNA hybrids exist in the nucleus and mitochondria but not in the cytosol, except in viral infection
We sought to test if RNA:DNA hybrids are present in the cytosol of human cancer cell lines
To analyze if RNA:DNA hybrids contribute to the PicoGreen signals in the cytosol, A549 cells were treated with RNase H, which degrades RNA in RNA: DNA hybrids [31], prior to staining with PicoGreen
Summary
RNA:DNA hybrids exist in the nucleus and mitochondria but not in the cytosol, except in viral infection. Results: RNA:DNA hybrids exist in the cytosol of various human cells and are mediated by RNA polymerase III (Pol III), which regulates the microRNA machinery. RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. We reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. We identified the presence of cytosolic RNA:DNA hybrids in immortalized and transformed human tumor cells. Chemical inhibition of Pol III abrogated the presence of cytosolic RNA:DNA hybrids in cells. We demonstrate the constitutive presence of cytosolic RNA:DNA hybrids in a variety of cell lines, and this accumulation depends on Pol III, at least in A549 lung carcinoma cells
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