Lost in translation? New findings suggest a critical role for cytosine methylation in tRNAs
While cytosine methylation in the DNA context will continue to be an important marker for epigenetic discoveries, current trends in the cytosine methylation game indicate diverse and critical roles of the modification in the RNA context. Much attention has been paid recently to findings in mRNA methylation, suggesting an important role in regulation of the transcriptome. New findings by Tuorto et al. now implicate cytosine methylation as an important player in the life and death of tRNAs.
Though cytosine methylation has been previously identified in tRNAs, no methods have been available to investigate its role in tRNA metabolism, let alone any greater biological context. In findings published recently in Nature Structural and Molecular Biology, researchers at the German Cancer Research Center (DKFZ) employed a broad arsenal of methods to paint a detailed picture of tRNA cytosine methylation. This study was built upon the foundation of earlier findings that pinpointed the Dnmt2 and NSun2 methyltransferases as primary players in tRNA methylation. It was observed that a knockout of either of these enzymes still resulted in viable offspring in the mouse model. However, when engineering a double knockout (DKO) of both enzymes, the researchers observed a frequency of homozygous DKO offspring lower than expected from Mendelian inheritance, suggesting a novel and strong genetic interaction between the two mutations.
The researchers observed several phenotypic changes in the surviving DKO offspring, generally demonstrating smaller size, lighter coloration, and incomplete skeletal ossification when compared to control litters. Interestingly, the DKO offspring also exhibited strong reductions in brown adipose tissue. This finding was solidified when fibroblasts from the DKO offspring were established to gauge their ability to differentiate into adipose cells in vitro, where they demonstrated a significantly diminished differentiation rate than the fibroblasts established from wild-type or single-knockouts (SKOs) of either enzyme.
In order to determine whether these observations were due to tRNA metabolism or changes in mRNA and gene expression, the researchers performed genome-wide mRNA expression profiling from the wild-type, SKO, and DKO genotypes, and observed that no transcripts demonstrated consistent changes between any of the genotypes. This finding suggested that the two methyltransferases can control proliferation events independent of any mRNA regulatory mechanisms. Through northern blot analysis, the team demonstrated that tRNA abundances in the DKOs were consistently significantly lower than SKO or wild-type genotypes, suggesting that the two methyltransferases work in concert to stabilize several key tRNAs. Finally, the researchers monitored the incorporation of labeled amino acids in cellular protein extracts from all genotypes, and observed distinct reductions in incorporation in the DKO cells when compared to the SKO or wild-type cells. The finding was corroborated when polysome profiles were observed: DKO cells and embryos both demonstrated a significant reduction in transcripts associated with polysomal ribosomes in comparison to SKOs or wild-type cells and embryos, suggesting a critical role of tRNA cytosine methylation in promoting normal translation events.
When considered together with recent discoveries in mRNA methylation patterns, these findings place cytosine methylation in prominent regulatory roles at each step in the flow of biological information. It is likely that as the sensitivity of methylation detection and mapping improve, more of the complex feedback loops between cellular events, gene products, and genes will become more clear.
Tuorto et al. (2012) RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat. Struc. Molec. Biol. doi:10.1038/nsmb.2357 Advance online publication (12 August 2012).