Dual-utility NLS drives RNF169-dependent DNA damage responses

An, Liwei; Jiang, Yiyang; Ng, Howin H. W.; Man, Ellen P.S.; Chen, Jie; Khoo, Ui‐Soon; Gong, Qingguo; Huen, Michael S.Y.

doi:10.1073/pnas.1616602114

Cited by 55 publications

(56 citation statements)

References 52 publications

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“…S3D) 64 . Taken together, these results indicate that DYRK1A expression and activity are required for efficient 53BP1 recruitment to DSBs within one hour, which contrasts with the reported role for RNF169 in antagonizing 53BP1 recruitment to promote HR and SSA 50,52 .…”

Section: Resolution Of 53bp1 Foci Was Measured By Quantifying the Deccontrasting

confidence: 77%

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

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Poss

Ebmeier

et al. 2018

Preprint

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Loss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known.Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus. MAIN BODY

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Section: Resolution Of 53bp1 Foci Was Measured By Quantifying the Deccontrasting

confidence: 77%

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

Guard

Poss

Ebmeier

et al. 2018

Preprint

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“…Further proteomic studies of the RNF169-DYRK1A complex in the cells before and after DNA damage will help to identify such factor. Our analysis of DYRK1A detected interaction with USP7, a ubiquitin-specific protease that has been recently shown to bind directly to RNF169 and to increase the stability of 53BP1, RNF169 and RNF168 (An et al, 2017; Liu et al, 2016; Yim et al, 2017; Zhu et al, 2015). Although disruption of the DYRK1A phosphorylation sites in RNF169 did not influence its interaction with USP7, the role of USP7 in the DYRK1A-RNF169 mediated regulation of 53BP1 should be further investigated.…”

Section: Discussionmentioning

confidence: 82%

“…Both the RNF169-AA and RNF169-DD mutants were able to bind polyubiquitin chains similar to the wild type RNF169, further supporting our conclusion that S368 and S403 sites do not play a role in RNF169’s accumulation at the DSB sites (Figure S3E). Furthermore, mutations of these sites did not affect the interaction with ubiquitin-specific protease USP7 that has been shown to be important for RNF169 function in DNA repair (An et al, 2017) (Figure S3F). Therefore, our data presented above support the conclusion that the intact S368 and S403 sites in RNF169, together with the DYRK1A kinase activity, are required for RNF169 to fully exert its ability to limit the recruitment of 53BP1 at the DSB sites after γ-radiation, through a mechanism unrelated to recognition of the ubiquitylated chromatin or USP7 binding by RNF169.…”

Section: Resultsmentioning

confidence: 99%

“…Further bioinformatic analysis of the 51 DYRK1A-binding proteins revealed a complex network of interactions of factors involved in different cellular processes with notable enrichment of the mRNA processing, transcription and DNA damage response functional categories (Figures 1D and E). Interestingly, recent proteomic analysis of RNF169 also identified DYRK1A as one of the most enriched interacting proteins (An et al, 2017). RNF169 is a RING-domain ubiquitin-binding protein that plays a role in the DNA repair signaling by regulating 53BP1, a scaffold protein that plays a key role in the choice of the double strand break (DSB) DNA repair by promoting non-homologous end joining (NHEJ) and inhibiting homologous recombination (HR) (Chen et al, 2012; Panier and Boulton, 2014; Poulsen et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

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DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Menon¹,

Ananthapadmanabhan²,

Swanson

et al. 2018

Preprint

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1Human DYRK1A gene encoding Dual-specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is a dosage-2 dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, 3 the function and regulation of this important protein kinase are not fully understood. Here we report proteomic 4 analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break 5 (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-6 binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-7 homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous 8 recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting 9 the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 10 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of 11 DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased 12 the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the 13 role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human 14 and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was 15 independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through 16 several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, 17 and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given 18 evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant 19 of the DNA damaging therapy response. 20 21

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“…The MS analysis of the protein band specific to PRLH1 RNA revealed that RNF169 specifically bound to PRLH1 with the highest and most significant score among 10 detected proteins ( Fig 4B and Appendix Table S1). RNF169 is an E3 ubiquitin ligase that has been reported as a negative regulator of the ubiquitin-dependent DNA damage signaling cascade and is involved in HR-mediated DNA repair [11,48,49].…”

Section: Prlh1 Specifically Binds To the Dna Repair Protein Rnf169mentioning

confidence: 99%

An LTR retrotransposon‐derived lnc RNA interacts with RNF 169 to promote homologous recombination

Deng

Wang

et al. 2019

EMBO Reports

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LTR retrotransposons are abundant repetitive elements in the human genome, but their functions remain poorly understood. Here, we report the function and regulatory mechanism of an ERV‐9 LTR retrotransposon‐derived lncRNA called p53‐regulated lncRNA for homologous recombination (HR) repair 1 (PRLH1) in human cells. PRLH1 is highly expressed in p53‐mutated hepatocellular carcinoma (HCC) samples and promotes cell proliferation in p53‐mutated HCC cells, and its transcription is promoted by NF‐Y and suppressed by p53. Mechanistically, PRLH1 specifically binds to an uncharacterized domain of RNF169 through two GCUUCA boxes in its 5′ terminal region to form a DNA repair complex that supplants 53BP1 at double‐strand break (DSB) sites and then promotes the initiation of HR repair. Notably, PRLH1 is essential for the stabilization of RNF169, acting as an RNA platform to recruit and assemble HR protein factors. This study characterizes PRLH1 as a novel HR‐promoting factor and provides new insights into the function and mechanism of LTR retrotransposon‐derived lncRNAs.

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Dual-utility NLS drives RNF169-dependent DNA damage responses

Cited by 55 publications

References 52 publications

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

An LTR retrotransposon‐derived lnc RNA interacts with RNF 169 to promote homologous recombination

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