TY - CHAP

T1 - Methods for Determination of 2′-O-Me in RNA

AU - Birkedal, Ulf

AU - Krogh, Nicolai

AU - Andersen, Kasper Langebjerg

AU - Nielsen, Henrik

PY - 2016/7/30

Y1 - 2016/7/30

N2 - Ribose methylation is one of the most abundant RNA modifications and is found in all kingdoms of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. The most abundant mechanism of ribose methylation is found in rRNA of Archaea and Eukarya where a methyltransferase (fibrillarin) use sRNA (Archaea) or box C/D snoRNA (Eukarya) as guide RNAs to specify the site of modification. The general function of these modifications is to promote ribosome biogenesis, in particular folding of the ribosomal RNA. Furthermore, some modifications affect the fidelity of translation. The function of individual modifications has in many cases remained elusive, because genetic deletion of the modification has a weak phenotype or no phenotype at all. Another problem is that methods for mapping modifications and quantitating the fraction of RNA molecules modified in a population until recently remained poorly developed. Here, we review the methods that have been used to study 2′-O-Me in RNA starting with the original approach employing in vivo isotope labeling followed by paper chromatography. The next generation of methods typically addressed one nucleotide at a time and was mostly based on primer extension. Finally, more recent mass spectrometry and high-throughput sequencing methods hold promise to reveal a new biology of this widespread type of nucleotide modification.

AB - Ribose methylation is one of the most abundant RNA modifications and is found in all kingdoms of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. The most abundant mechanism of ribose methylation is found in rRNA of Archaea and Eukarya where a methyltransferase (fibrillarin) use sRNA (Archaea) or box C/D snoRNA (Eukarya) as guide RNAs to specify the site of modification. The general function of these modifications is to promote ribosome biogenesis, in particular folding of the ribosomal RNA. Furthermore, some modifications affect the fidelity of translation. The function of individual modifications has in many cases remained elusive, because genetic deletion of the modification has a weak phenotype or no phenotype at all. Another problem is that methods for mapping modifications and quantitating the fraction of RNA molecules modified in a population until recently remained poorly developed. Here, we review the methods that have been used to study 2′-O-Me in RNA starting with the original approach employing in vivo isotope labeling followed by paper chromatography. The next generation of methods typically addressed one nucleotide at a time and was mostly based on primer extension. Finally, more recent mass spectrometry and high-throughput sequencing methods hold promise to reveal a new biology of this widespread type of nucleotide modification.

U2 - 10.1007/978-3-319-34175-0_8

DO - 10.1007/978-3-319-34175-0_8

M3 - Book chapter

SN - 978-3-319-34173-6

T3 - RNA Technologies

SP - 187

EP - 205

BT - Modified Nucleic Acids in Biology and Medicine

A2 - Jurga , Stefan

A2 - Erdmann , Volker A.

A2 - Barciszewski, Jan

PB - Springer Science+Business Media

CY - switzerland

ER -