Loss of CARM1 is linked to reduced HuR function in replicative senescence
- Lijun Pang†1,
- Haiyan Tian†1,
- Na Chang1,
- Jie Yi1,
- Lixiang Xue1,
- Bin Jiang1,
- Myriam Gorospe2,
- Xiaowei Zhang1Email author and
- Wengong Wang1Email author
© Pang et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 2 July 2013
Published: 9 July 2013
The co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR. However, the functional impact of this modification is not fully understood. Here, we investigated the influence of HuR methylation by CARM1 upon the turnover of HuR target mRNAs encoding senescence-regulatory proteins.
Changing the methylation status of HuR in HeLa cells by either silencing CARM1 or mutating the major methylation site (R217K) greatly diminished the effect of HuR in regulating the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16. Although knockdown of CARM1 or HuR individually influenced the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16, joint knockdown of both CARM1 and HuR did not show further effect. Methylation by CARM1 enhanced the association of HuR with the 3′UTR of p16 mRNA, but not with the 3′UTR of cyclin A, cyclin B1, c-fos, or SIRT1 mRNAs. In senescent human diploid fibroblasts (HDFs), reduced CARM1 was accompanied by reduced HuR methylation. In addition, knockdown of CARM1 or mutation of the major methylation site of HuR in HDF markedly impaired the ability of HuR to regulate the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16 as well to maintain a proliferative phenotype.
CARM1 represses replicative senescence by methylating HuR and thereby enhancing HuR’s ability to regulate the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs.
RNA binding protein HuR, the ubiquitously expressed member of Hu RNA binding proteins , is functionally involved in the regulation of mRNA stabilization, translation, and export [2–4]. Among the cellular events that are influenced by HuR and its target mRNAs is the process of replicative senescence. Several studies have implicated HuR in regulating the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs, as well as the nuclear export of HuR mRNA during replicative senescence [4–8].
Although the molecular events controlling HuR function are not fully understood, the cytoplasmic presence and post-translational modification (e.g., phosphorylation and methylation) of HuR are particularly important [2, 9–11]. For example, the cell cycle checkpoint kinase Chk2 has been shown to interact with HuR and phosphorylate HuR at residues S88, S100, and T118; phosphrylation at S100 seems to be important for the dissociation of the HuR-SIRT1 mRNA complex in response to oxidative stress . The cyclin-dependent kinase 1 (CDK1) phosphorylates the HuR hinge region at serine 202 and leads to enhanced HuR association with 14-3-3, thereby retaining HuR in the nucleus . Additionally, studies by Doller and coworkers identified HuR as a substrate for both PKCα and PKCδ. The phosphorylation of HuR at S221 by both PKCα and PKCδ is also linked to HuR shuttling as well as the stabilization of COX-2 mRNA . These findings underscore functional links between HuR and the aforementioned signaling cascades, which regulate HuR shuttling or its binding to target mRNAs.
Besides phosphorylation, methylation is another important post-translational modification of HuR. The co-activator associated arginine methyltransferase 1 (CARM1) catalyses the methylation of HuR and HuD in vitro and in vivo[13, 14]. CARM1 has been reported to methylate the histones (e.g.,H3,H4) and transcriptional factors [e.g., p53,hormone-activated nuclear receptor (NR), etc.] at arginine residues, thereby activating gene transcription [15–18]. Methylation by CARM1 enhances the function of HuD and HuR in stabilizing TNF-α and SIRT1 mRNAs [14, 19], respectively. The R217 present within the HuR hinge region is identified as the major methylation site by CARM1 . It was proposed that the interaction of HuR nuclear ligands SETα/β, pp32, and APRIL with the HuR hinge region regulates HuR shuttling . However, thus far, the functional impact of CARM1-mediated HuR methylation on replicative senescence and the precise mechanism underlying remain largely unexplored.
In this study, by using two approaches to mimic the hypomethylation of HuR, knockdown of CARM1 and mutation of HuR at the major methylation site (R217), we have investigated the functional impact of the CARM1-mediated HuR methylation and the mechanisms underlying in replicative senescence. Our results indicate that the methylation by CARM1 is critical for HuR to regulate the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16 in replicative senescence.
Methylation by CARM1 enhances the effect of HuR in regulating the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs
We next asked if CARM1 functions through methylating HuR. HeLa cells were transfected with HuR siRNA, CARM1 siRNA or both siRNAs, and 48 h later, the levels of cyclin A, cyclin B1, c-fos, SIRT1, and p16 were assessed by Western blot analysis. As anticipated, knockdown of HuR (lanes 2) or CARM1 (lanes 3) individually reduced the levels of cyclin A, cyclin B1, c-fos, and SIRT1, and increased the levels of p16 (Figure 1E). However, joint knockdown of HuR and CARM1 (lanes 4) was not more effective than individual knockdown of HuR or CARM1 (Figure 1E). In sum, by methylating HuR, CARM1 is able to regulate the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16.
Methylation by CARM1 enhances the association of HuR with p16 3′UTR, but not cyclin A, cyclin B1, c-fos, and SIRT1 3′UTR
Besides its localization, the interaction of HuR with target mRNAs influences its ability to regulate mRNA turnover or translation. To further test the influence of CARM1-mediated methylation on the interaction of HuR with RNA, biotinylated fragments of cyclin A, cyclin B1, c-fos, SIRT1, and p16 3′UTRs and cytoplasmic extracts described in Figure 3A and 3B were used for pull-down and RNP IP assays, as previously described . As shown in Figure 3C and 3D, knockdown of CARM1 had no influence on the association of HuR with the 3′UTRs of cyclin A, cyclin B1, c-fos, and SIRT1 mRNAs. In addition, the association of flag-HuR (W) and flag-HuRΔ (M) with the 3′UTR of cyclin A, cyclin B1, c-fos, and SIRT1 was comparable (Figure 3E and 3F). However, changing HuR methylation either by silencing CARM1 (Figure 3C and 3D) or by mutating the HuR methylation site (Figure 3E and 3F) markedly lowered the association of HuR with p16 3′UTR. In a previous study, HuR and AUF1 were found to bind with the p16 3′UTR and destabilize p16 mRNA cooperatively . To address whether methylation of HuR by CARM1 influences the association of AUF1 with p16 3′UTR, the lysates described in Figure 3C and 3E were used for pull-down and RNP IP assays. As shown in Figure 3G and 3H (left), in cells with silenced CARM1, the association of AUF1 with p16 3′UTR was markedly reduced. Moreover, in cells expressing flag-HuRΔ (M), the association of AUF1 with the p16 3′UTR was substantially weaker than that observed from cells expressing flag-HuR (W) (Figure 3G and 3H, right). These results suggest that methylation by CARM1 enhances the association of HuR with the p16 3′UTR, and in turn, enhances the association of AUF1 with the p16 3′UTR.
CARM1- HuR regulatory process impacts on replicative senescence
The present study provides novel insight into the regulation of HuR function in replicative senescence. By modulating the HuR methylation status, we gained evidence to support the view that methylation by CARM1 critically affects HuR’s ability to regulate the levels of cyclin A, cyclin B1, c-fos, SIRT1, and p16 as well as the process of cell senescence (Figures 1, 2, 4, 5, and 6).
Reduced HuR levels and cytoplasmic concentration have been linked to the lower expression of cyclin A, cyclin B1, c-fos, and SIRT1 and the increased expression of p16 in replicative senescence [5–7]. Thus, signaling events that regulate the relative levels of HuR in the nucleus and the cytoplasm may also involve in the process of cell aging . Although the mechanism controlling HuR shuttling has not been fully elucidated, the hinge region of HuR is of great importance for its nuclear localization . Because the Arg217 localizes at the hinge region, we hypothesized that the methylation of HuR by CARM1 may elevate the expression of HuR targets by increasing the cytoplasmic presence of HuR. However, lowering HuR methylation by either silencing CARM1 or mutating the major methylation site had no effect on the shuttling of HuR (Figure 3A, B). It is also possible that methylation of HuR affects its RNA-binding affinity because methylation may affect the interaction of HuR with the hinge-binding proteins, which could in turn modulate RNA binding . Indeed, ithas been reported that methylation by CARM1 could enhance the association of HuR with SIRT1 3′UTR during the differentiation of human embryonic stem cells (hESC) . Here, loss of HuR methylation either by silencing CARM1 or by mutating the major methylation site did not influence the association of HuR with the 3′UTRs of SIRT1, cyclin A, cyclin B1 or c-fos. However, methylation of HuR by CARM1 enhanced the association of HuR to p16 3′UTR (Figure 3C-H). Therefore, whether methylation by CARM1 influences the binding affinity of HuR may not only depend on the methylation status of HuR and the mRNAs targeting by HuR, but also on the type and physiologic state of the cell in which the HuR-mRNA interaction occurs.
In addition to HuR and HuD, methylation of poly(A)-binding protein 1, CA150, the SAP49, SmB, and the U1 small nuclear RNP-specific protein U1C has been implicated in the process of mRNA stabilization as well as the 5′ splice site selection of the pre-mRNA splicing [20–22]. Methylation of hnRNP A2, the nuclear poly (A)-binding protein PABPN1, and the poly (U)-binding proteins Sam68 and SLM by PRMT1 influences their function and proper localization [23–25]. Because both the expression and methylation status of HuR significantly decline with replicative senescence (Figure 4), it is challenging to evaluate the contribution of CARM1-mediated methylation to the reduction of HuR methylation in senescent cells. However, the evidence presented here (Figures 5 and 6) suggests that the CARM1- HuR regulatory process do contribute to the regulation of genes associated with replicative senescence. Although the links between human aging and replicative senescence are not fully understood, senescent cells accumulate with advancing age and actively contribute to physiologic and pathologic changes of aging. Our finding that CARM1 contributes to the alterations of cyclin A, cyclin B1, c-fos, SIRT1, and p16 in replicative senescence warrants a careful look at CARM1 in other models of aging. In light of the fact that HuR is also an important regulator of cell death [26, 27], cell differentiation [19, 28, 29], and human cancer [10, 30–32], we postulate that CARM1-HuR regulatory process may impact upon these processes as well.
CARM1-mediated protein methylation enhances the function of HuR upon the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16. Methylation by CARM1 did not influence HuR subcellular distribution or association with the 3′UTRs of cyclin A, cyclin B1, c-fos, and SIRT1 mRNAs, but it enhanced the association of HuR with the p16 3′UTR. By methylating HuR, CARM1 critically regulates replicative senescence.
Cell culture, fluorescence-activated cell sorting (FACS) analysis, and senescence-associated β-galactosidase (SA-β-gal) activity
Early-passage (Young, ~25-27 population doublings[pdl]), late-passage (Senescent, ~60 pdl) human diploid 2BS fibroblasts (National Institute of Biological Products, Beijing, China), and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, at 37 C in 5% CO2. Fluorescence-activated cell sorting (FACS) analysis, and senescence-associated β-galactosidase (SA-β-gal) activity were performed as described previously .
Constructs and transfection
For the construction of vectors expressing flag-HuR, full-length coding region of HuR was amplified by PCR using flag-tagged primer GGAATTCATGGACTACAAGGACGACGATGACAAGTCTAATGGTTATGAA and primer GCTCTAGATTATTTGTGGGACTTGTTGG and inserted between EcoRI sites of pcDNA 3.1 vector (Clontech). The flag-HuR bearing an arginine-to-lysine mutation on residue 217 were generated using QuikChange® II Site-Directed Mutagenesis Kit (Stratagene) and inserted between EcoR and Xbal sites of pcDNA 3.1 (Clontech). For construction of vectors expressing CARM1 or control shRNAs, oligonucleotides corresponding to siRNA targeting CARM1 (CAGCTCT ACATGGAGCAGT), HuR (AAGAG GCAAUUACCAGUUUCA), or a control shRNA (AAGTGTAGTAGATC ACCAGGC) were inserted between the hind III and BgIIIsites in pSuper.retro (Oligoengine) vector following the manufacturer’s instructions.
All plasmid or siRNA transfection in HeLa cells were performed using lipofectamine 2000 (for plasmids) or oligofectamine (for siRNAs) (Invitrogen) following the manufacturer’s instructions. Cells were collected 48 h after transfection for further analysis. To establish lines stably expressing flag-HuR, flag-HuRΔ, or CARM1 shRNA, early-passage (~25 pdl) 2BS cells were transfected with a vector expressing flag-HuR, flag-HuRΔ, CARM1 shRNA, or with the respective control vectors by lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions, selected by G418 (300 μg/ml, Invitrogen) for 3–4 weeks, and maintained in medium supplemented with 50 μg/ml G418.
Preparation of cell fractions, immunoprecipitation (IP) assays, and Western blot analysis
Whole-cell lysates as well as cytoplamic and nuclear extracts were prepared as described previously . IP assays were performed using 50 μg of whole-cell lysates and 1 μg of HuR or flag antibody (M2). For Western blot analysis, lysates were size fractionated by SDS-PAGE and transferred onto poly-vinylidene difluoride (PVDF) membranes. Monoclonal antibodies recognizing HuR, HDAC1, p16, c-fos, SIRT1, β-tubulin, and GAPDH were from Santa Cruz Biotechnologies (Santa Cruz, Calif.). Mouse monoclonal flag antibody (M2) was from Sigma. Mouse monoclonal mono/dimethyl arginine antibody (M/DMA) was from Abcam. After secondary antibody incubation, signals were detected by Super Signal West Pico Chemiluminescent Substrate (Pierce) following the manufacturer’s instruction.
RNA-protein interaction assays and UV crosslink RNP IP assays
cDNA was used as a template for PCR amplification to generate the 3′UTR of different HuR target transcripts. All 5′ primers contained the T7 promoter sequence CCAAGCTTCTAATACGACTCACTATAGGGAGA-. To prepare the 3′-UTR fragments for cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs, primers (T7) CCAGAGACATAAATCTGTAAC and GGTAACAAATTTCTGGTTTATTTC for cyclin A 3′UTR, primers (T7) CTTGTAAACTTGAGTTGGAGT and TTTTTTTTTTTTGTATTTGAG for cyclin B1 3′UTR, primers (T7) GCAATGAGC CTTCCTCTGAC and CATTCAACTTAAATGCTTTTATTG for c-fos 3′UTR, primers (T7) AACTATCCATCAAACAAA and TATCCAGTCATTAAACAGT for SIRT1 3′UTR, and primers TTGGTCCCTCTTGATTAT and GTGATGTCTGGCTGTTTC for p16 3′UTR were used, respectively. For biotin pull-down assays, PCR-amplified DNA was used as template to transcribe biotinylated RNA by using T7 RNA polymerase in the presence of biotin-UTP, as described . One microgram of purified biotinylated transcripts were incubated with 100 μg of cytoplasmic extracts for 30 min at room temperature. Complexes were isolated with paramagnetic streptavidin-conjugated Dynabeads (Dynal, Oslo), and the pull-down material was analyzed by Western blotting.
For cross-linking of RNP IP complexes, cells were exposed to UVC (400 mJ/cm2) and whole-cell lysates prepared for immunoprecipitation using monoclonal anti-HuR and polyclonal anti-AUF1 antibodies, as described . The transcripts present in the RNP complexes were analyzed by real-time qPCR.
RNA isolation, real-time qPCR, and mRNA half-life measurement
Total cellular RNA was prepared using RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. For real-time qPCR against GAPDH analysis to detect cyclin A, cyclin B1, c-fos, SIRT1, p16, and β-actin transcripts, primers TTGGTCCCTCTTGATTAT and GTGATGTCTGGCTGTTTC for cyclin A, primers GCACTTTCCTCCTTCTCA and CGATGTGGCATACTTGTT for cyclin B1, primers CGAAGGGAAAGGAATAAGATG and TGAGCTGCCAGGATGAACT for c-fos, primers TAGGCGGCTTGATGGTAATC and TCATCCTCCATGGGTTCTTC for SIRT1, primers GAAGGTCCCTCAGACATCCCC and CCCTGTAGGACCTTCGGTGAC for p16, and primers GTGGACATCCGCAAAGAC and AAAGGGTGTAACGCAACTAA for β-actin were used, respectively.
To measure the half-life of endogenous mRNAs, the expression of mRNA was shut off by adding actinomycin D (2 μg/ml) into the cell culture medium, whole cellular RNA was prepared at times indicated and subjected to real-time qPCR. Data were plotted as the mean ± SD from 3 independent experiments and the half-lives were calculated as previously described .
This work was supported by the grants 8123008, 81070274, 30973147, and 81170320 from National Science Foundation of China; Grant B07001 from the Ministry of Education of People’s Republic of China (111 project); Grant 20090001110059 from the Research Fund for the Doctoral Program of higher Education of China. MG was supported by the National Institute on Aging-IRP, National Institutes of health. We thank J. Shay for generously providing us the IDH4 cells.
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