Sequence- and structure-selective mRNA m⁵C methylation by NSUN6 in animals

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• Messenger RNA (mRNA) N6-methyladenosine (m⁵C) modifications regulate crucial cellular processes like mRNA mobility, metabolism, and translation.
• Two distinct types of m⁵C sites exist in mRNAs: Type I, methylated by NSUN2, and Type II, with unknown biogenesis.
• Type II m⁵C sites are characterized by a downstream UCCA motif and predicted location in hairpin loops.

Purpose of the Study:

• To identify the methyltransferase responsible for Type II m⁵C mRNA modification.
• To elucidate the RNA sequence and structural determinants of NSUN6-mediated mRNA methylation.
• To investigate the functional consequences of NSUN6-targeted mRNA m⁵C methylation.

Main Methods:

• Utilized RNA secondary structure prediction and miCLIP (methylation-specific crosslinking and immunoprecipitation).
• Performed high-throughput mutagenesis analysis to determine RNA sequence and structural features.
• Developed an NSUN6-RNA structural model and engineered an NSUN6 variant.

Main Results:

• Identified NSUN6 as the methyltransferase responsible for Type II m⁵C mRNA sites.
• Determined the specific RNA sequence and structural requirements for NSUN6 binding and methylation.
• Created an NSUN6 variant with altered substrate specificity (reduced tRNA methylation, retained mRNA methylation).
• Observed a weak negative correlation between m⁵C methylation and translation efficiency.

Conclusions:

• mRNA m⁵C modification is regulated by a sophisticated two-enzyme system involving NSUN2 and NSUN6.
• NSUN6 specifically targets Type II m⁵C sites in mRNA, distinct from its known tRNA methylation.
• The developed protein-RNA structure analysis strategy can be applied to study other RNA modification writers and their substrates.