Methylcytosine – It’s not just for DNA anymore. Despite being almost exclusively associated with DNA for decades, methylcytosine (5mC) has recently been shown to be an important base modification in RNA as well. Exploding interest in the RNA version of 5mC, what it does, and how it works, has been sparked by a sharp uptick in studies published over the last couple of years. Here are a few prime examples of RNA methylation in action:
- Tuorto et al., 2012 showed that RNA methylation stabilized mouse tRNAs and promoted protein synthesis
- Squires et al., 2012 demonstrated that many RNA species, including mRNAs contain 5-methylcytosine
- Hussein et al., 2013 found RNA methylation managed the processing of vault RNAs (vtRNAs) into various small regulating RNAs
Bisulfite Comes Up Big for Methylation Detection in RNA
Clearly RNA methylation is a big factor in the epigenetic landscape, but a major obstacle to better understanding it, is the lack of available analysis techniques. Any approach needs to account for a couple of things – Stabilizing the 5mC mark for analysis, and the site-specificity, or base-pair resolution, of the method. To the development team at Zymo Research, bisulfite conversion seemed like the perfect tool for the job.
Bisulfite conversion, the Rolling Stones of DNA methylation analysis techniques, has been rocking longer than most of us care to remember, but until just recently hadn’t been applied to RNA, when a couple of papers from German researchers (Schaefer et al., 2009 and Schaefer et al., 2010) showed that it was possible to adapt bisulfite sequencing to investigate RNA methylation. These papers worked mostly with transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but opened the door for methylation research powerhouses, like Zymo Research, to develop the first tools that would be useful and familiar to scientists delving into the RNA methylation space.
EZ RNA Methylation Kit™ Makes the Scene
Capitalizing on their vast bisulfite expertise, the Zymo R&D team designed the first kit ever for looking at RNA methylation, the EZ RNA Methylation™ Kit. This new kit looks and functions much like the classic DNA bisulfite kits you know and love, but it’s tailored specifically for RNA methylation analysis.
Zymo researcher Michiko Suwoto gave us some insights on how the new technique was optimized to investigate RNA methylation. First, they dialed back the conversion reaction. “The bisulfite conversion conditions required are not as harsh for RNA as those for DNA, reflecting both the nature of the molecule and its susceptibility to the conversion.” Says Suwoto.
The other key difference is in the clean-up and desulphonation steps, which are critical if you want to actually be able to use your converted product in downstream analysis. According to Suwoto, “Desulphonation is normally done at high pH, which will destroy RNA molecules. By optimizing this step of the procedure specifically for RNA, we have enabled RNA to be used with bisulfite conversion”.
Even though it’s only been on the market for a short time, the EZ RNA Methylation™ kit is already making a splash by showing up in a recent journal article (Amort et al, 2013) where scientists discovered that RNA methylation altered the function and chromatin binding of long non-coding RNAs (lncRNAs) XIST and HOTAIR.
To learn more about adopting bisulfite conversion for your own RNA methylation studies, check out the EZ RNA Methylation™ Kit at the Zymo Research website.