Repression of PUM1-mediated mRNA decay activates translesion synthesis after DNA damage

ABSTRACT

Biological roles of Pumilio1 (PUM1) in ubiquitous cells remain unclear. Here we identify 48 degrading target mRNAs by combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs. Further analysis revealed that cells respond to DNA damage by inhibiting PUM1-mediated mRNA decay to activate translesion synthesis (46/50).

Pumilio 1 (PUM1) is an RNA-binding protein that recognizes a single 5ʹ-UGUAHAUA-3ʹ (H = A, U, or C) sequence, named Pumilio Response Element (PRE), and degrades RNAs containing PREs.¹ Although PUM1 is known to have functional roles in development and neuronal activity,^2,3 biological roles of PUM1 expressed in most of somatic cells are unclear. Given the fundamental lack of knowledge with degrading target mRNAs of PUM1, we developed an approach to determine mRNAs that both bind to and are degraded by PUM1.⁴

To identify mRNAs bound to PUM1, we performed RNA immunoprecipitation sequencing (RIP-seq) and found that PUM1 binds to 3,097 mRNAs. Next, we performed 5ʹ-bromouridine (BrU) immunoprecipitation chase sequencing (BRIC-seq). BRIC-seq determines genome-wide RNA stabilities by chasing chronological decrease of BrU-labeled RNAs under physiologically undisturbed conditions.^5,6 We found that 101 mRNAs were stabilized by PUM1 knockdown compared to control cells. By extracting intersections of RIP-seq and BRIC-seq results, we identified 48 mRNAs that were both bound to PUM1 and degraded by PUM1 (Figure 1(a)).

Figure 1.

A scheme to identify PUM1 targets and biological role of PUM1-mediated mRNA decay. (a) A strategy to determine RNAs that are bound to Pumilio1 (PUM1) and degraded by PUM1. PUM1-binding mRNAs were identified by RNA immunoprecipitation sequencing (RIP-seq), whereas mRNAs degraded by PUM1 were determined by 5ʹ-bromouridine (BrU) immunoprecipitation chase sequencing (BRIC-seq). (b) a model of PUM1 function in activation of translesion synthesis (TLS) after DNA damage

Next, we searched for biological conditions that modulate PUM1-mediated mRNA decay by developing in silico screening scheme. In this scheme, we assumed that if stimuli influence PUM1-mediated mRNA decay, then those stimuli will cause inversely correlated changes between PUM1 mRNAs abundance and the abundance of its degradation targets. For example, inhibitory stimulus of PUM1-mediated mRNA decay decreases PUM1 abundance and increases the abundance of the degradation targets. For this in silico screening, we collected 491 RNA-seq data obtained under various biological conditions from Gene Expression Omnibus. Our in silico screening found that anti-cancer drugs, such as camptothecin and cisplatin, were potential inhibitors of PUM1-mediated mRNA decay. As these anti-cancer drugs cause DNA damage, we hypothesized that PUM1 may have functional role in DNA damage.

To confirm the results of in silico screening, we analyzed the amounts of PUM1 and its target mRNAs in cisplatin-treated cells. We found that cisplatin decreased both mRNA and protein levels of PUM1, whereas cisplatin stabilized and increased PUM1 degrading targets in HeLa Tet-Off cells. Notably, those increased PUM1 degrading targets contained genes related to DNA repair after DNA damage (Proliferating cell nuclear antigen (PCNA) and Ubiquitin Conjugating Enzyme E2 A (UBE2A)). Moreover, these inverse correlations are also found in HCT116 and A549 cells.

Cisplatin induces apoptosis by introducing DNA lesions which lead to stall of DNA polymerase during DNA replication.⁷ A major DNA damage tolerance process is translesion synthesis (TLS) that involves DNA synthesis by TLS polymerases, such as Pol η, to bypass the damaged template.⁸ Because UBE2A and PCNA are necessary to activate TLS, we thought that cells may activate TLS by suppressing PUM1-mediated mRNA decay.

During activation of TLS, UBE2A monoubiquitinates PCNA.⁹ We found that cisplatin increased the abundance of UBE2A, PCNA, and monoubiquitinated PCNA in HCT116 cells. Moreover, cells overexpressing PUM1 did not show increase of UBE2A and PCNA, and monoubiquitination of PCNA, indicating that inhibition of PUM1-mediated mRNA decay increases monoubiquitination of PCNA.

Considering that PUM1 overexpression inhibits the monoubiquitination of PCNA, we predicted that PUM1 overexpression would impair DNA synthesis by inhibiting TLS. To quantify DNA synthesis, we performed BrdU incorporation assay. HCT116 cells reduced DNA synthesis with cisplatin treatment (31% synthesis compared to the normal state). Importantly, cells overexpressing PUM1 in cisplatin-treated condition reduced DNA synthesis to 21%, which was significantly lower than the normal cells. These data suggested that PUM1 overexpression impaired DNA synthesis by inhibiting TLS in response to DNA damage.

Inhibition of DNA replication in DNA-damaged condition induces cell apoptosis. We hypothesized that cells overexpressing PUM1 reduces cell viability in the cisplatin-treated condition. Compared with control HCT116 cells exposed to cisplatin, PUM1-overexpressing cell viability was significantly lower (69% compared to 80%). Taken together, our results indicate that cells resist to the cellular toxicity of DNA-damaging reagents by suppressing PUM1-mediated mRNA decay (Figure 1(b)).

Previous studies showed that UBE2A activates TLS, but the activation mechanism was unknown.¹⁰ Here, we revealed that cisplatin decreased PUM1 and inhibited PUM1-mediated mRNA decay. The inhibition of PUM1 function increased UBE2A and PCNA by stabilizing their mRNAs, leading to increase monoubiquitinated PCNA. We also showed that we can inhibit TLS and reduce cell viability to DNA-damaging reagents by overexpressing PUM1. Thus, inhibition of PUM1-mediated mRNA decay increases the effectiveness of such chemotherapeutics.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.