m7G-MaP-seq

m7G-MaP-seq detects internal N7-methylguanosine (m7G) RNA modifications at nucleotide resolution using mutational profiling sequencing.

Key Features:

• High-throughput detection: Identifies m7G modifications at single-nucleotide resolution across RNA species.
• Mutational profiling technique: Converts m7G-modified positions to abasic sites by sodium borohydride reduction, records them as cDNA mutations during reverse transcription, and detects them by sequencing.
• Statistical analysis: Compares mutation rates between reduced and control samples and assesses significance with log likelihood ratio tests across replicates to account for sequencing and alignment errors.
• Efficiency in rRNA detection: Demonstrates mutational rates up to 25% for known m7G sites in ribosomal RNA (rRNA).
• Discovery of novel modification: Mapped a previously uncharacterized, evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA.
• Broad organismal application: Identified m7G modifications in tRNAs from budding yeast, humans, and Arabidopsis and showed these modifications occur prior to tRNA splicing.
• Specificity and limitations: Does not detect internal m7G modifications in small RNAs such as miRNA, snoRNA, or sRNA (including human Let-7e), and extensive mRNA sequencing from E. coli or yeast revealed no internal m7G.

Scientific Applications:

• Research on RNA modifications: Enables investigation of the roles and disease implications of internal N7-methylguanosine in RNA.
• Evolutionary studies: Supports mapping of conserved m7G modifications to study evolutionary conservation of RNA modification sites.
• RNA biology: Facilitates analysis of the functional significance of m7G modifications in tRNAs and rRNAs.

Methodology:

Mutation rates are compared between reduced and control samples and statistical significance is assessed using log likelihood ratio tests across replicates to account for sequencing and alignment errors.