Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m⁶A modification

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms.
• The lncRNA pncRNA-D is induced by irradiation and interacts with RNA-binding protein TLS/FUS to inhibit cyclin D1 (CCND1) expression.
• The role of RNA modifications in the pncRNA-D-TLS interaction and subsequent gene regulation remains unclear.

Purpose of the Study:

• To investigate the role of N6-methyladenosine (m6A) modification in the interaction between pncRNA-D and TLS/FUS.
• To elucidate how m6A modification of pncRNA-D affects CCND1 gene expression and cell cycle progression.
• To identify the m6A reader protein involved in the pncRNA-D-TLS interaction.

Main Methods:

• Cell culture (HeLa cells) and induction of pncRNA-D via osmotic stress.
• Knockdown of m6A modification enzyme METTL3 and m6A reader protein YTHDC1 using siRNA.
• RNA immunoprecipitation (RIP), RNA pulldown assays, and CRISPR/Cas9-mediated gene editing.
• Cell cycle analysis and cell growth assays.

Main Results:

• Osmotic stress induced pncRNA-D and reduced its m6A methylation, alongside TLS arginine methylation.
• Knockdown of METTL3 or YTHDC1 enhanced the pncRNA-D-TLS interaction.
• YTHDC1 was identified as the m6A reader for pncRNA-D, mediating the inhibitory effect on the TLS-pncRNA-D interaction.
• Reduced m6A modification of pncRNA-D led to cell cycle arrest at G0/G1 phase and suppressed cell growth.

Conclusions:

• m6A modification of pncRNA-D is crucial for regulating its interaction with TLS/FUS.
• This m6A-dependent regulation impacts CCND1 expression and controls cell cycle progression.
• YTHDC1 acts as a key mediator in the m6A-driven regulatory pathway involving pncRNA-D.