May 13, 2017

High-throughput and site-specific identification of 2'-O-methylation sites using ribose oxidation sequencing (RibOxi-seq)

RNA
Yinzhou ZhuGordon G Carmichael

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.

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Mentioned in this Paper

Small Nuclear RNA
MRNA Maturation
Dimethoxymethane
Protein Methylation
Sequence Determinations, RNA
Binding Sites, Antibody
Nucleic Acid Sequencing
Biologic Development
Archaea
Ribosomal RNA

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