NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation

Abstract

A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5′-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5′UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence.

NSUN2-Mediated m5C Methylation and METTL3/METTL14-Mediated m6A Methylation Cooperatively Enhance p21 Translation

“…All plasmids were transfected using lipofectamine 2000 (Invitrogen) and cells were collected 48–72 h after transfection for further analysis. SA‐β‐galactosidase staining was performed as described previously [Tang et al, ; Cai et al, ].…”

“…The p21 3′UTR10 mutants (3′UTRCm [bearing C2079G], 3′UTR10m1 [bearing A1986U], 3′UTR10m2 [bearing A2037U], 3′UTR10m3 [bearing A2044U], 3′UTR10m4 [bearing A2061U], 3′UTR10m5 [bearing A2044U and A2061U] were prepared by overlapping PCR). The p27 5′UTR and SHC 5′UTR fragments were described previously [Tang et al, ; Cai et al, ].…”

“…The tRNA methyltransferase NSun2 (Misu) is a typical RNA methyltransferase catalyzing the formation of m5C in coding and no‐coding RNAs [Hussain et al, ; Tang et al, ; Xing et al, ; Cai et al, ]. Methylation of mRNAs by NSUN2 is found to regulate the translation or turnover of mRNAs encoding CDK1, TP53, SHC, p16 (CDKN2A), and p27 (CDKN1B), thus affecting the expression of these genes in processes such as cell proliferation, oxidative stress, and replicative senescence [Hussain et al, ; Tang et al, ; Xing et al, ; Cai et al, ]. METTL3 (methyltransferase like 3), METTL14 (methyltransferase‐like 14), and WTAP (Wilm's tumor 1‐associated protein) form a methyltransferase complex, which catalyzes m6A formation [Liu et al, ; Ping et al, ].…”

RNA modifications and structures cooperate to guide RNA–protein interactions

“…Expression of the m 5 C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) is tightly regulated during the cell cycle and its overexpression delays replicative senescence through methylation of Cyclin-dependent kinase 1 (CDK1) and the CDK inhibitor 1B (p27 KIP1 ) transcripts. Deposition of m 5 C in the CDK1 3′UTR enhances its translation, while m 5 C in the 5′UTR of p27 KIP1 represses its translation, resulting in increased cellular proliferation 56, 57 . Deposition of hm 5 C by methylcytosine dioxygenases 58 in mRNA correlates with higher association with polyribosomes in Drosophila melanogaster 59 .…”

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

“…Given the relevance of cellular senescence to mammalian aging, we initiated a systemic investigation as to whether RNA modification regulates RNA abundance during human aging. Specifically, we focused on the m6A RNA modification because it is the most abundant internal modification observed in eukaryotic mRNA, which can affect mRNA splicing and nuclear export; in the cytoplasm, m6A mRNAs are recognized by the RNA‐binding protein (RBP) that will mobilize them to processing (P) bodies, where mRNA stability and translation are modulated (Fu, Dominissini, Rechavi & He, 2014; Licht & Jantsch, 2016; Tang et al., 2015). …”