More Methylation in RNA!?! RNA Continues to Surprise…
While DNA methylation is an extensively-studied epigenetic modification, relatively little is known about the methylation of cytosine bases in RNA. Although the functional consequences of RNA modifications are not clear, RNA molecules are known to be extensively modified. In fact, there are over 100 reported modifications to RNA, which is even more than are currently known for DNA. Recently, Hussain et al. developed a new technique for site-specific detection of 5-methylcytosine in RNA, which they coined Methylation individual-nucleotide-resolution CrossLinking and ImmunoPrecipitation, or miCLIP. The authors employed their miCLIP method to explore the targets of NSun2 and found that tRNAs, mRNAs, and ncRNAs were all targets of this protein. They confirmed their findings using other available techniques to detect cytosine methylation in RNA, such as bisulfite conversion and 5-azacytidine-mediated immunoprecipitation, validating the miCLIP method.
This new method cleverly utilizes a mutant NSun2 to covalently link the protein to target RNA molecules. The mechanism by which the enzyme methylates cytosine requires the formation of a covalent bond between cysteine 321 in NSun2 and a pyrimidine base in RNA. The liberation of the RNA from the protein requires a second cysteine at position 271, and mutation of this conserved cysteine to alanine prevents the release of the RNA from NSun2. Ultimately, this mutation creates a protein that becomes covalently attached to RNA methylation target sites, allowing these sites to be identified by immunoprecipitation of NSun2.
While exploring the targets of NSun2 using the miCLIP method, the authors detected approximately 300 mRNAs containing 5-methylcytosine, indicating that NSun2-mediated methylation of coding RNA is a fairly rare event. A 2012 study suggested that methylation of mRNA may play a role in increasing the stability of the modified RNA, leading the authors to test this hypothesis by using RNAi to knock down NSun2 expression. They did not find any significant changes in mRNA levels when NSun2 levels were depleted. Furthermore, the authors investigated RNA levels in human skin fibroblasts that were either homozygous or heterozygous for a loss-of-function NSUN2 mutation, and found that of the 312 identified mRNAs identified by miCLIP, greater than 90% of their levels remained largely unchanged. Hussain et al. therefore concluded that 5-methylcytosine in mRNA does not confer additional stability to the modified RNA molecules.
In addition to the mRNA and tRNA targets, the authors found that a class non-coding RNAs called Vault RNAs (vtRNAs) were also methylated by NSun2. vtRNAs are non-coding RNAs that form a ribonucleoprotein Vault complex that has no known function at this time. Interestingly, a recent report found that the quantity of vtRNAs is greatly increased during neural differentiation and additional reports found that NSun2 deficiency causes neurodevelopment issues in humans. Taken together, the data suggest that methylation of vtRNAs might play an important role in development and differentiation of the nervous system.
vtRNAs are also known substrates of the Dicer complex and can be processed into small regulatory RNAs (svRNAs); therefore the authors speculated that 5-methylcytosine could affect the processing of vtRNAs. This hypothesis was tested by incubating both unmethylated and methylated forms of a specific vtRNA, vtRNA1.1, with cell lysates and quantifying svRNA processing by qPCR. They found one svRNA, svRNA4, was more prevalent when the RNA was methylated, indicating that RNA methylation can regulate vtRNA processing. In addition, the authors found that the differential svRNA4 processing affected expression of several target mRNAs, including CACNG7 and CACNG8.
After taking all of these results into account, the authors suggest that that the NSun2-mediated methylation of vtRNA may act as a molecular switch for the expression of mRNAs that are regulated by svRNAs.
Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J, & Frye M (2013). NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs. Cell reports, 4 (2), 255-61 PMID: 23871666