m⁶ A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Alternative splicing (AS) coupled with nonsense-mediated mRNA decay (NMD) is a key mechanism for regulating gene expression.
• Understanding the full scope of AS-NMD targets is crucial for deciphering cellular regulatory networks.

Purpose of the Study:

• To identify mRNAs regulated by AS-NMD in an organism.
• To investigate the role of METT-10 and m6A modification in AS-NMD regulation of S-adenosyl-L-methionine (SAM) synthetase genes.

Main Methods:

• Long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient Caenorhabditis elegans mutant strain.
• Direct RNA sequencing and machine learning for m6A modification analysis.
• In vitro methylation assays.

Main Results:

• Identified 259 high-confidence AS-NMD genes, including sams-3 and sams-4.
• Demonstrated that SAM synthetase activity autoregulates sams gene expression via AS-NMD in a negative feedback loop.
• Showed METT-10 is essential for AS-NMD regulation in vivo and methylates the 3' splice site AG dinucleotide in vitro, with m6A modification confirmed on endogenous sams mRNAs.

Conclusions:

• Homeostasis of SAM synthetase in C. elegans is maintained by AS regulation.
• m6A modification at the 3' splice site of sams genes by METT-10 is a critical regulatory mechanism.