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. 2005 Feb;6(2):158-64.
doi: 10.1038/sj.embor.7400346.

Increased stability of the p16 mRNA with replicative senescence

Wengong Wang  1 , Jennifer L Martindale, Xiaoling Yang, Francis J Chrest, Myriam Gorospe
Affiliations

Increased stability of the p16 mRNA with replicative senescence

Wengong Wang et al. EMBO Rep. 2005 Feb.

Abstract

Expression of p16(INK4a) is elevated during ageing and replicative senescence. Here, we report the presence of an instability determinant within the 3'-untranslated region (UTR) of the p16 messenger RNA in WI-38 human diploid fibroblasts. The p16 3'UTR was found to be a specific target of AUF1, an RNA-binding protein implicated in promoting mRNA decay. Both AUF1 levels and AUF1-p16 mRNA associations were strikingly more abundant in early-passage than late-passage fibroblast cultures. Moreover, short interfering RNA-based reductions in AUF1 levels increased the stability of p16 3'UTR-containing transcripts, elevated the expression of p16 and accentuated the senescence phenotype. Together, our findings show that p16 mRNA turnover decreases during replicative senescence and that the instability-conferring region is located within the 3'UTR of p16, as well as identifying AUF1 as a critical mediator of these regulatory events.

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Figures

Figure 1
Figure 1
The p16 3′UTR confers instability to chimeric transcripts in young WI-38 cells. (A) Northern blot analysis of p16, cyclin A and p21 expression in young (Y, pdl 25) and senescent (S, pdl 60) WI-38 HDFs. (B) Schematic representation of the p16 mRNA, depicting the 3′UTR ARE (black), and the constructs prepared for chimeric RNA analysis. (C) Young and senescent WI-38 cells were transiently transfected with the constructs shown in (B) (1 μg each) along with the tet repressor plasmid (20 μg), and then cultured for an additional 20 h. Following addition of Dox (1 μg/ml), northern blotting was performed to assess the half-life of each transcript (supplementary information online).
Figure 2
Figure 2
Binding of proteins to p16 transcripts. (A) REMSA analyses were performed using nuclear or cytoplasmic protein lysates (10 μg each) obtained from young and senescent WI-38 cells. RNA probes encompassed the CR or the 3′UTR. Supershift REMSA was performed with antibodies recognizing AUF1, HuR, p38 or p27. (B) Whole-cell lysates (400 μg) were prepared from young HDFs, and endogenous target transcripts were detected by RT–PCR assay of the corresponding IP material; PCR products corresponding to GAPDH mRNA (which bound IP materials at background levels, thus serving as loading control) and p16 mRNA were visualized on agarose gels. (C) Western blot of AUF1 levels in total (10 μg), cytoplasmic (40 μg) and nuclear (10 μg) lysates prepared from either young or senescent WI-38 cells. AUF1 isoforms (p37, p40, p42 and p45) are indicated. Cytoplasm-specific β-tubulin and nucleus-specific HDAC1 (histone deacetylase 1) signals showed the quality and loading of the cytoplasmic and nuclear preparations, respectively. (D) Pull-down assay (supplementary information online) using biotinylated fragments A–C (left) to detect bound cytoplasmic AUF1 by western blotting (right). A 5 μg portion of whole-cell lysate (Lys.) and biotinylated GAPDH (negative control, Neg.) was included.
Figure 3
Figure 3
p16 3′UTR confers instability to chimeric transcripts in lung carcinoma cells. (A) Following transient transfection of H2 cells (constitutively expressing the tet repressor) with the constructs depicted in Fig 1B (1 μg each), Dox was added, RNA was collected at the times shown and the rates of clearance of each transcript (mRNA half-life) were monitored by northern blotting, as described (supplementary information online). (B) Western blot of AUF1 expression in H2 cells 3 days after transfection with plasmid pSILENCER-AUF15 (AUF1 siRNA), expressing an siRNA that targeted all four AUF1 isoforms, or control plasmid pSILENCER (lane C); β-actin, loading control. (C) At 3 days after transfection of H2 cells as explained in (B), plasmids expressing either EGFP(V) or EGFP-3′UTR(S) were transfected and the stability of the chimeric RNAs was tested (explained in (A)) in both the C and the AUF1 siRNA treatment groups.
Figure 4
Figure 4
AUF1 modulates WI-38 senescent phenotype and p16 expression. Following a sequential co-transfection of WI-38 cells with an EGFP-expressing plasmid along with either a control plasmid (lane C) or an AUF1 siRNA-expressing vector, EGFP-positive cells were sorted by flow cytometry and used to assess the expression of AUF1 (A) and additional genes (B) by either western blotting or RT–PCR, as well as β-galactosidase-positive cells (C). (D) WI-38 HDFs were co-transfected with an EGFP-expressing plasmid and each of four isoform-specific AUF1 expression vectors and AUF1 levels (top, dots indicate individual overexpressed AUF1 isoforms) and [3H]thymidine incorporation (graph) were assessed. Vector, insertless plasmid. Graphs represent the means+s.e.m. of 3–5 independent assessments.
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