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.

SUBMITTER: Yoneda R 

PROVIDER: S-EPMC7186179 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature