Mapping RNA to DNA with MARGI

Over the past decade, it has become much easier, quicker, and cheaper to sequence the human genome. Organizations, like 23&me, have even made it possible for anyone to understand about their genome and any downstream effects their genes have on their health. However, there is still a lot about DNA interactions that we know relatively very little about. RNA, known as the intermediate between DNA and protein,  actually has a huge amount of regulatory power over your genes but is very difficult to study. Researchers at the University of California San Diego have recently developed a technique to overcome this problem that has the potential to map all RNA to DNA interactions in a single experiment. This could lead to a better understanding the regulatory roles of RNA and the possibility of preventing diseases like cancer or even autism.

Keywords: DNA, RNA, transcription, epigenetics.

The Central Dogma of molecular biology explains the flow of genetic data within a biological system. DNA, the information of life, which is carefully contained inside the cell nucleus, is transcribed into mRNA and taken to parts of the cell that are translated into proteins. Proteins are responsible for signalling, scaffolding, and catalyzing metabolic reactions, along with many other functions within a cell.

Most of the human genome, though sequenced a multitude of times, is not fully understood. Even though we know the order of the nucleotides within the DNA, it does not mean that we know what proteins with which they correlate. In fact, only about 2% of the whole human genome contains information about encoding proteins, the rest are hypothesized to be involved in regulation. This suggests that most gene products are, in fact, RNA that will bind to DNA and determine what is and is not transcribed into a protein product. For example:

RNA inhibits creation of apoptotic protein. Apoptosis is programmed cell death. Under normal circumstances, we don’t want our cells to die but in disease states such as cancer, it would be better for the cell to abort mission and prevent the spread of the mutation. However, in some cancers there is an increase in siRNA, short interfering RNA (green), that bind to DNA (blue). If the transcription protein (red) cannot bind, then cell degradation cannot happen. It would be great to be able to understand what RNA sequences enhance cancer to be able to stop them from binding.

In general, scientists are interested in knowing which genes are ultimately responsible in self regulation of DNA through RNA products.

Existing methods to study RNA to DNA interactions analyze one RNA molecule at a time, involving hundreds of unstable RNA strands and multiple years to analyze all of the interactions. However, using a new technology called MARGI (MApping RNA Genome Interactions) an entire set of DNA to RNA interactions could be studied in one experiment and then analyzed in only a few weeks!

The MARGI technique works by first pooling fragments of RNA with fragments of DNA together with the hopes that interacting DNA and RNA could bind. A designed linker is then added to connect the interacting pairs of RNA and DNA. This linker is used to fish out interacting strands from the rest of the fragments which are sequenced. The sequenced results from a MARGI experiment yields in a map of RNA to DNA and could potentially yield a lot of insight into when RNA will bind and modify DNA transcription.

Diagram of MARGI technique. When RNA (green) and DNA (blue) fragments interact, a linker comes in and covalently binds them together. The linker has a tag (star) on it so the interacting strands can be pulled out of the fragment pool for sequencing. Both the DNA and RNA are sequenced to determine which genes are regulated by each RNA product. Returning to our apoptotic gene above, MARGI would be able to identify the siRNA that prevents cell degradation in cancerous states.

These researchers were able to identify some known RNA-DNA interactions, but their data suggests that there may be many more RNA structures that can associate to DNA and affect gene products. Surprisingly, many of the RNA-DNA interactions had a significant amount of overlap, but with that said, their library may contain a lot of false positives. For their control, they mixed RNA from fruit flies and DNA from human and got about 2% interaction suggesting that this new technique is not very selective.

Nevertheless, MARGI is the first of its kind to be able to map an entire genome’s worth of RNA-DNA interactions and is a big step towards understanding how RNA helps regulate gene products and affects transcription. This technique especially can be applied to understand epigenetics, the study of how protein levels are altered without a change in DNA sequence, which has been implicated with cancer, autism, and mental health. Understanding how RNA is able to interact with DNA might shed some light on how to prevent and even reverse diseased and stressed states.

If you want to hear more about RNA, check out the RNA Symposium at UMich tomorrow (Friday, 31 March 2017). Michigan Science Writers, myself included, will be covering the event live on their blog and on Twitter. Follow the conversation with #umichRNA!

Sarah Kearns is a first year in the Chemical Biology Doctoral Program at the University of Michigan. Currently, she is doing research rotations to find a long-term lab environment ideally focusing on enzyme structure-function relationships for drug development applications. You can find her on Twitter (@annotated_sci), LinkedIn, or at her website Annotated Science.

Image Sources | Header: Wikimedia Commons. Figures 1 & 2: made by Sarah Kearns.