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Catalytic crosslinking-based methods for enzyme-specified profiling of RNA ribonucleotide modifications
- Hussain, Shobbir
- Methods 2019 v.156 pp. 60-65
- Escherichia coli, RNA, chemical bonding, crosslinking, cytosine, methylation, methyltransferases, ribonucleotides
- Well over a hundred types of naturally occurring covalent modifications can be made to ribonucleotides in RNA molecules. Moreover, several types of such modifications are each known to be catalysed by multiple enzymes which largely appear to modify distinct sites within the cellular RNA. In order to aid functional investigations of such multi-enzyme RNA modification types in particular, it is important to determine which enzyme is responsible for catalysing modification at each site. Two methods, Aza-IP and methylation-iCLIP, were developed and used to map genome-wide locations of methyl-5-cytosine (m5C) RNA modifications inherently in an enzyme specific context. Though the methods are quite distinct, both rely on capturing catalytic intermediates of RNA m5C methyltransferases in a state where the cytosine undergoing methylation is covalently crosslinked to the enzyme. More recently the fundamental methylation-iCLIP principle has also been applied to map methyl-2-adenosine sites catalysed by the E. coli RlmN methylsynthase. Here I describe the ideas on which the two basic methods hinge, and summarise what has been achieved by them thus far. I also discuss whether and how such principles may be further exploited for profiling of other RNA modification types, such as methyl-5-uridine and pseudouridine.