An unorthodox partnership in DNA repair pathway choice

Abstract

Complex regulatory circuits determine whether DNA double‐strand breaks (DSBs) are repaired by nonhomologous end‐joining (NHEJ) or homology‐directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng et al [1] report that a novel p53‐suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR‐mediated DSB repair and proliferation of p53‐deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

The two principal mechanisms for repairing DSBs, NHEJ and HDR, actively compete for resolving these cytotoxic lesions 2, 3. Improperly executed DSB repair can be detrimental to genome integrity and ultimately undermine cell and organismal fitness. Accordingly, the choice between NHEJ and HDR pathways for faithful repair of individual DSBs is governed by a regulatory framework of remarkable and ever‐growing intricacy 4. Chromatin ubiquitylation by the E3 ubiquitin ligases RNF8 and RNF168 centrally influences DSB repair pathway utilization by recruiting key repair factors, in particular NHEJ‐promoting components including the 53BP1‐RIF1‐Shieldin and BRCA1‐A complexes, to the break sites 4, 5, 6. Reflecting its physiological importance, the activity of the RNF8‐RNF168 pathway is controlled by multifaceted regulatory cues 5.

In this issue, Deng et al 1 describe an unorthodox novel regulatory principle operating in the context of the RNF8‐RNF168 signaling axis to impact DSB repair pathway choice. By analyzing RNA‐seq data from large cohorts of hepatocellular carcinomas (HCCs), they first identified an uncharacterized primate‐specific long terminal repeat (LTR) retrotransposon‐derived long noncoding RNA (lncRNA), PRLH1 (p53‐regulated lncRNA for HR repair 1), whose expression is transcriptionally repressed by p53 and, consequently, strongly upregulated in p53‐mutated HCCs. Moreover, PRLH1 expression correlates positively with cell proliferation in p53‐deficient, but not wild‐type, backgrounds. A proteomic search for PRLH1‐interacting proteins revealed the ubiquitin ligase RNF169, an RNF168 paralog recognizing the RNF168‐generated histone ubiquitylation mark at DSB sites. This modification is also read by 53BP1 albeit with lower affinity, thus enabling RNF169 to antagonize 53BP1 accumulation at DSBs and thereby shift the equilibrium of DSB repair pathway utilization toward HDR 6. The PRLH1‐RNF169 interaction entails two conserved GCUUCA boxes in PRLH1 and a functionally uncharacterized region in RNF169 that is not shared by RNF168; indeed, no binding of PRLH1 to RNF168 could be observed. Similar to RNF169, high levels of PRLH1 expression led to diminished 53BP1 recruitment to DSB sites and augmented HDR efficiency, and vice versa. Mechanistically, Deng et al provide evidence that PRLH1 stabilizes RNF169 protein, which could plausibly explain its ability to stimulate RNF169 accumulation at DSB sites and HDR. Importantly, while PRLH1 binds several cellular proteins, its impact on 53BP1 association with DSBs, HDR efficiency, and cell proliferation appears to be largely RNF169‐dependent. By stabilizing RNF169, PRLH1 thus emerges as an RNA‐based component in the arsenal of competing cellular NHEJ‐ and HDR‐promoting activities that modulate protein interactions with DSB sites to specify repair pathway choice (Fig 1).

Figure 1. The p53‐regulated lncRNA PRLH1 influences DSB repair pathway choice by stabilizing the E3 ubiquitin ligase RNF169.

In normal cells, p53 represses expression of the LTR retrotransposon‐derived lncRNA PRLH1 mediated by the NF‐Y transcription factor (left panel). Functional inactivation of p53 leads to transcriptional induction of PRLH1 that directly interacts with and stabilizes the E3 ubiquitin ligase RNF169 (right panel). As RNF169 recognizes the same DSB‐associated ubiquitin (Ub) mark as the pro‐NHEJ factor 53BP1 but has higher affinity for it, the PRLH1‐dependent increase in RNF169 abundance effectively diminishes 53BP1 association with DSB sites, thereby shifting the balance between competing DSB repair mechanisms toward HDR usage.

The new findings by Deng et al add PRLH1 to a small but growing list of lncRNAs regulating cellular responses to DSBs 7. Mechanistically, however, PRLH1 appears to differ from other lncRNAs involved in DSB repair by exerting its function directly at the level of protein stabilization, rather than acting through the more conventional route of regulating gene expression transcriptionally or post‐transcriptionally. Whether complex formation with RNF169 enables PRLH1 to impact repair factor assembly at DSB sites more directly than by merely stabilizing RNF169 remains to be seen. Considering the large number of LTR retrotransposons 8, it is conceivable that other lncRNAs might regulate DNA repair and genome integrity maintenance processes via similar mechanisms; further studies of such lncRNA–protein interplay are clearly warranted. In the absence of a canonical RNA‐binding motif in RNF169, it will be of interest to more precisely establish how it is bound and stabilized by PRLH1, whether the PRLH1‐RNF169 interaction is subject to regulatory control or subcellular compartmentalization, and whether this has functional relevance beyond DSB repair. In this regard, while the identification of a lncRNA targeting RNF169 underscores its functional importance in cells, it remains unclear whether the ability of RNF169 to restrain 53BP1 accumulation at DSB sites represents its primary in vivo role, given that this does not require its E3 ligase activity 6. As PRLH1 enhances the proliferation of HCC cells lacking functional p53 via RNF169, it will be important to address whether or not this effect is attributable to its HDR‐stimulating function.

Previous work suggested a complex but predominantly HDR‐suppressive function of p53 in regulating DSB repair 9. The findings by Deng et al raise the possibility that increased RNF169 expression and HDR usage mediated by de‐repressed PRLH1 transcription could provide a selective growth advantage to p53‐mutated cancer cells, a notion that may offer new therapeutic opportunities if further substantiated. Future studies should help clarifying whether such a p53‐regulated PRLH1‐RNF169 axis is operational in malignancies other than HCC and whether PRLH1 and/or RNF169 could thus be candidate novel treatment targets or biomarkers in tumors harboring mutant p53.

EMBO Reports (2019) 20: e49105