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

Abstract

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

Emerging Contribution of PancRNAs in Cancer

“…Furthermore, pncRNA-D is susceptible to m6A methylation and its methylation was dramatically decreased after osmotic stress or irradiation in HeLa cells [24]. Notably, it has been reported that the inhibition of methylation enhances the stability of pncRNA-D, thus promoting its interaction with FUS/TLS [24]. Therefore, the methylation process seems to ensure the control of cyclin D1 levels through two alternative ways.…”

“…Accordingly, the inhibition of pncRNA-D by siRNA strategy or RNAseT1 treatment impairs the binding of FUS/TLS to the CCND1 promoter, further supporting the dependence of FUS/TLS from this specific ncRNA in the regulation of CCND1. Intriguingly, the methylation status of FUS/TLS as well as of pncRNA-D can affect the functionality of this molecular mechanism [24]. FUS/TLS contains two glycine-arginine rich (RGG) domains in its C-terminal region, which are directly involved in the binding to pncRNA-D [25].…”

“…Methylation of arginine R-476 disrupts the pncRNA-D-FUS/TLS interaction, thus leading to cyclin D1 upregulation through the rescue of CBP/p300 histone acetyl-transferase (HAT) activity [26]. Furthermore, pncRNA-D is susceptible to m6A methylation and its methylation was dramatically decreased after osmotic stress or irradiation in HeLa cells [24]. Notably, it has been reported that the inhibition of methylation enhances the stability of pncRNA-D, thus promoting its interaction with FUS/TLS [24].…”

Jack of all trades? The versatility of RNA in DNA double-strand break repair

“…However, it is now understood that the m 6 A modification is required for the DDR to UV damage, providing proof of principle that RNA modifications can function in the DDR. A role for m 6 A at DSB sites is yet to be established, although m 6 A levels have been found to be altered in response to DSBs [ 127 , 135 ]. m 6 A and METTL3 were found to be elevated in glioma stem-like cells in response to IR, and silencing of METTL3 reduced DSB repair and enhanced the sensitivity to IR in these cells [ 135 ].…”

“…m 6 A and METTL3 were found to be elevated in glioma stem-like cells in response to IR, and silencing of METTL3 reduced DSB repair and enhanced the sensitivity to IR in these cells [ 135 ]. Moreover, the lncRNA pncRNA-D m 6 A modification levels were found to be reduced in response to IR or osmotic stress, which can induce DSBs, suggesting m 6 A can be modulated in response to DSBs [ 127 ].…”