Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry

Lim, Jung Hyun; Giri, Pankaj K.; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie Alyssa; Chen, Yuling; Rabadán, Raúl; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

doi:10.1016/j.cell.2017.03.043

Cited by 61 publications

(85 citation statements)

References 45 publications

(69 reference statements)

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“…This enrichment of G mutations is lost in cNHEJ-deficient cells, while the relative pattern of G mutations (to A, C, T) is not affected, suggesting that cNHEJ affects AID targeting to 5′Sμ, but not the processing of the U:G mismatch. The relative abundance of sense vs. antisense transcripts (42), and the preference of APOBEC family enzymes to deaminate the lagging strand during DNA replication (39,43), could all contribute to this strand bias.…”

Section: Discussionmentioning

confidence: 99%

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Crowe

Shao

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs ( ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in , but not , B cells. Meanwhile, the residual joints from cells and the efficient Sµ-Sγ1 junctions from B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

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Section: Discussionmentioning

confidence: 99%

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Crowe

Shao

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Exosomes were isolated using a Total Exosome Isolation Kit (System Biosciences, Palo Alto, CA, USA) [34,35]. The isolated exosome pellet was resuspended in 1/10 of the original volume in sterile water for subsequent detection and analysis [36][37][38].…”

Section: Methodsmentioning

confidence: 99%

Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis

Zhang

Zhao²,

et al. 2020

BMC Genomics

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Background: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed "pseudomales" who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring.Results: To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylationrelated and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish. Conclusions: These signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed "pseudomales" in C. semilaevis.

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“…Consistent with this model, the frequency of reads with mutations also decreased in DNA-PKcs 5A/5A B cells. The bias of APOBEC family enzymes to deaminate the lagging strand during DNA replication (53, 56) and the relative abundance of sense vs anti-sense transcripts (57) could all contribute to this strand bias.…”

Section: Discussionmentioning

confidence: 99%

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Crowe

Wang

Shao

et al. 2020

Preprint

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18The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the 19 large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) 20 factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with 21 different isotypes by joining two DNA double-strand breaks at different switching regions via 22 the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair 23 phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and recruits and activates DNA-PK. 24 DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at 25 both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation 26 sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs 5A 27 mouse model carrying an alanine substitution at the T2609 cluster, here we show that loss of 28 T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR 29 junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocation, 30 excess end-resection, and preferential usage of micro-homologyall signs of the alternative 31 end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation 32 in promoting cNHEJ repair pathway choice during CSR. 33 34 Key points 35 Loss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.36 37 58 recombination. Correspondingly, DNA-PKcs -/-(null) or Artemis -/mice are born of normal size at 59 the expected ratio but develop severe combined immunodeficiency (SCID) (4-6). Patients with 60 4hypomorphic mutations in DNA-PKcs or Artemis also develop SCID (12, 13). Mature B cells 61 undergo CSR, which replace the initially expressed IgM constant region with another 62 downstream constant region encoding a different isotype, to generate antibodies with different 63 effector functions. The cNHEJ pathway plays an important role in CSR. But in cells lacking a 64 core cNHEJ factor (e.g., Lig4 or Xrcc4), up to 25-50% of CSR can be achieved by the alternative 65 end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. 66 Consistent with DNA-PKcs and Artemis being dispensable for direct end-ligation, DNA-PKcs -/-67 B cells only have moderate defects in CSR (14, 15). Nevertheless, in recent high-throughput 68 sequence analyses, we found that CSR junctions recovered from DNA-PKcs -/-B cells contained 69 increased chromosomal translocations and extensive end-resection, and preferentially used MHs 70 (16), suggesting that the seemingly robust CSR achieved in DNA-PKcs -/cells is primarily 71 mediated by the Alt-EJ pathway like those in Lig4 -/or Xrcc4 -/-B cells (17, 18). Accordingly, 72 human patients with spontaneous mutations in DNA-PKcs show severe defects in both V(D)J 73 recombination and CSR and increased MH in the residual CSR junctions (12, 19). 74Active DNA-PK phosphorylates many substrates, among which, DNA-PKcs is the best-75 characterized...

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Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry

Cited by 61 publications

References 45 publications

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

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