What’s good for the goose is good for the gander, so why shouldn’t bisulfite sequencing be used to study RNA cytosine methylation? Until recently, the harsh reaction conditions required for bisulfite deamination were considered detrimental to RNA stability. However, Matthias Schaefer and colleagues developed an RNA bisulfite sequencing procedure that reproducibly and quantitatively detected cytosine methylation of tRNAs and rRNAs.
Covalent modifications are actually more prevalent and complex in RNA than in DNA and have been observed in rRNA, mRNA, miRNA, and tRNA. But relatively little is known about the functions of RNA covalent modifications because analytical methodologies have lagged behind those for DNA.
RNA Bisulfite Sequencing
To establish the RNA bisulfite sequencing protocol, Schaefer and co-workers analyzed two RNAs that are known to be methylated at specific sites Drosophila tRNAAsp and E. coli 16S rRNA. Low-temperature (60 C) bisulfite conversion was followed by reverse transcription of the RNA, PCR amplification, cloning, and sequencing.
The researchers optimized the amount of RNA and the temperature and time of bisulfite treatment to minimize RNA degradation and to efficiently deaminate unmethylated cytosines. Furthermore, bisulfite conversion in combination with deep sequencing was used to precisely quantify the methylation patterns of tRNA (Val 3) , tRNA (Val 4), and tRNA( Lys) in cDNA libraries from Drosophila embryos.
The investigators say that the identification and quantification of methylated cytosines with the new method could provide novel insights into the functions of RNA methylation. See for yourself at Nucleic Acids Research, December 2008.