METTL3 alters capping enzyme expression and its activity on ribosomal proteins

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• The 5' cap is crucial for mRNA function, maturation, and gene expression regulation.
• RNA guanylyltransferase and 5'-phosphatase (RNGTT) catalyzes 5' capping.
• N6-methyladenosine (m6A) is a prevalent RNA modification influencing mRNA fate and function.

Purpose of the Study:

• To investigate the regulatory role of m6A in RNGTT expression and activity.
• To determine if m6A modification affects RNGTT protein levels and mRNA capping efficiency.

Main Methods:

• Western blotting to assess RNGTT protein levels.
• RNA immunoprecipitation followed by sequencing (RIP-Seq) to identify m6A sites.
• Quantitative real-time PCR (qRT-PCR) to measure mRNA levels.
• Pathway analysis (e.g., mTOR, p70S6K) to understand downstream effects.

Main Results:

• The 3' untranslated region (3'UTR) of RNGTT mRNA is methylated by METTL3.
• Knockdown of METTL3 leads to decreased RNGTT protein expression.
• Inhibition of cytoplasmic capping dysregulates gene expression, particularly ribosomal protein and 5' terminal oligopyrimidine (TOP) mRNAs.
• METTL3 knockdown disrupts mTOR and p70S6K signaling pathways, reducing RPS6 mRNA capping, protein expression, and phosphorylation.

Conclusions:

• METTL3-mediated m6A modification of RNGTT mRNA is a novel regulatory mechanism for RNGTT expression.
• m6A-dependent regulation of RNGTT impacts cytoplasmic mRNA capping, affecting translation of specific mRNA subsets.
• This finding highlights a link between RNA epigenetic modifications and key cellular signaling pathways like mTOR.