Abstract
Ribose methylation (2′-O-methylation, 2′-OMe) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2′-O-methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3′-ends, followed by periodate oxidation of all molecules terminating in 2′,3′-OH groups. This allows only RNAs harboring 2′-OMe groups at their 3′-ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth.
Highlights
A great majority of 2′-O-methylations are directed by Box C/D snoRNAs, noncoding RNAs that guide the modification of target sites via complementary RNA sequences
Studies demonstrated that 2′-O-methylations on rRNAs are indispensable for ribosome biogenesis (Tollervey et al 1993); 2′-O-methylation has been shown to be present on tRNAs and has been implicated to be crucial in translational circuitries (Satoh et al 2000; Guy et al 2015)
Ribose methylated bases are found at mRNA caps and are involved in host pathogen responses (Daffis et al 2010; Rimbach et al 2015)
Summary
A great majority of 2′-O-methylations are directed by Box C/D snoRNAs, noncoding RNAs that guide the modification of target sites via complementary RNA sequences. This study identified over 400 sites, almost 300 more than what have been curated in human rRNAs (Lestrade and Weber 2006) It is unclear, how many of the novel sites are true positives, owing to an inherent high false-positive rate of primer extension. By randomly hydrolyzing RNA and performing next-generation sequencing at very high depth, there should be uniform coverage of 3′-end positions across regions of interest except at positions of 2′-O-methylation This method overall has much better specificity and accuracy, as it has successfully detected about the same number of sites in rRNAs as have been annotated. In order to address these issues, we have developed a 2′-O-methyl ribose-specific, high-throughput method, which relies on positive rather than negative signals, to detect 2′-O-methylation sites
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