Catalytic crosslinking-based methods for enzyme-specified profiling of RNA ribonucleotide modifications

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Over 100 types of RNA ribonucleotide modifications exist, often catalyzed by multiple enzymes targeting distinct sites.
• Identifying the specific enzyme responsible for each RNA modification is crucial for functional studies.

Purpose of the Study:

• To develop and describe enzyme-specific methods for mapping RNA modifications.
• To investigate the application of these methods for profiling various RNA modification types.

Main Methods:

• Development of Aza-IP and methylation-iCLIP techniques to map genome-wide RNA modifications.
• Both methods capture catalytic intermediates of RNA methyltransferases, crosslinking the enzyme to the modified cytosine.
• Adaptation of the methylation-iCLIP principle for mapping methyl-2-adenosine sites.

Main Results:

• Successfully mapped genome-wide locations of methyl-5-cytosine (m5C) modifications in an enzyme-specific context.
• Demonstrated the application of methylation-iCLIP for mapping methyl-2-adenosine sites catalyzed by E. coli RlmN methylsynthase.

Conclusions:

• Aza-IP and methylation-iCLIP are effective for enzyme-specific RNA modification mapping.
• The principles of these methods show potential for profiling other RNA modifications, including methyl-5-uridine and pseudouridine.