Identification of a selective polymerase enables detection of N(6)-methyladenosine in RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• N(6)-methyladenosine (m(6)A) is the most prevalent mRNA modification in eukaryotes.
• m(6)A plays crucial roles in various biological processes and human health.
• Precisely locating m(6)A modifications within RNA sequences remains a significant technical challenge.

Purpose of the Study:

• To develop a method for the precise localization and quantification of m(6)A modifications in RNA.
• To identify a specific enzyme capable of distinguishing between modified and unmodified adenosine residues.
• To determine the exact position of m(6)A in mammalian 28S ribosomal RNA (rRNA).

Main Methods:

• Screening and identification of a polymerase with reverse transcriptase activity from *Thermus thermophilus*.
• Characterization of the enzyme's selectivity for incorporating thymidine opposite unmodified adenosine over m(6)A.
• Application of the selective polymerase, in conjunction with a nonselective polymerase, for m(6)A mapping in synthetic and cellular RNAs.

Main Results:

• The identified *Thermus thermophilus* polymerase exhibits up to 18-fold selectivity for unmodified adenosine over m(6)A.
• The enzyme enables the location and quantification of m(6)A in synthetic RNAs via analysis of pausing bands.
• The study successfully determined the precise location of m(6)A in mammalian 28S rRNA to be at nucleotide 4190.

Conclusions:

• A novel enzymatic approach has been established for the precise mapping of m(6)A modifications.
• This method overcomes previous limitations in identifying specific m(6)A positions within RNA molecules.
• The determination of m(6)A at nucleotide 4190 in mammalian 28S rRNA provides critical insights into RNA regulation and function.