Mapping R-loops in the human genome
New method maps these three-stranded structures almost to the resolution of single letters of the DNA code.
Genes contain coded instructions for making proteins. When the cell needs to use a gene, molecular machinery assembles near the start of the gene in regions called promoters. Part of this machinery then reads along the gene, making a copy of the code in the form of a DNA-like molecule called RNA. These RNAs typically contain regions called exons, which carry the instructions, interspersed with spacer regions called introns. As RNAs are made they are ‘spliced’ to chop out the introns, leaving behind the final instructions.
Most DNA exists in a double helix shape with two connected DNA strands, but the regions near the start of genes often contain structures called R-loops. In these structures, one strand of the DNA partners up with a single strand of RNA, forcing the other strand to bulge out on its own. Their location at gene promoters indicates that R-loops could change the cell’s use of genes by impacting the machines that assemble near the start of genes. However, R-loops are not well understood. A major barrier to understanding the role of R-loops is that we do not know exactly where they are with respect to the start of genes.
Dumelie and Jaffrey now report a new method to map R-loops almost to the resolution of single letters of the DNA code — a method which they called bisDRIP-seq. The approach extends an existing technique called DRIP-seq, which uses antibodies to capture DNA sequences stuck to strands of RNA. It can find R-loops, but it cannot tell the difference between the loop itself and the DNA surrounding it. The new technique uses a chemical called bisulfite to alter the DNA letters. It only affects the loop of the R-loop because the RNA shields the other strand. Sequencing then pinpoints the modified letters, revealing the exact location of the loop.
For human cells grown in the laboratory, the technique found that R-loops form between the start of the gene and its first intron. Some genes do not have any introns, and in these cases, the R-loops extended deep into the code. Most human genes have only a small amount of DNA between the start site and the first intron, which may act to limit the effect of R-loops in these genes.
This new technique allows the high-resolution study of R-loops, and could help to reveal their role in regulating genes. Abnormal R-loops have already been linked to a small set of human diseases like fragile-X syndrome. As the tools to study R-loops improve, it is possible that scientists will make connections to other diseases. In time, improved understanding of these structures could lead to better diagnosis, and eventually treatment, for these conditions.
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