Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an ...RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.
The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show ...that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.
The degeneration of axon terminals before the soma, referred to as "dying back", is a feature of Parkinson's disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify ...protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to "dying back" pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics.
PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass ...spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing. One-third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder, and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.
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•MS-based phosphoproteomic profiling of PPM1D substrates in U2OS and HCT116 cells•PPM1D counteracts ATM in the cellular response to DNA double-strand breaks•PPM1D target sites localize to glutamic acid-rich regions with high intrinsic disorder•Kanadaptin is a putative DNA damage response factor regulated by ATM and PPM1D
Biochemistry; Molecular biology; Cancer; Proteomics.
Oncoproteins of the MYC family drive the development of numerous human tumours
. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II
.... MYC proteins can also coordinate transcription with DNA replication
and promote the repair of transcription-associated DNA damage
, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks
. MYC multimerization is triggered in a HUWE1
and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.
PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and ...prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.
A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often ...poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.
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•Tailor-made E3s afford PCNA polyubiquitylation with alternative linkages•Alternative linkages confer distinct fates to polyubiquitylated PCNA•Linkage reprogramming is generalizable to other monoubiquitylated target proteins
Wegmann et al. have designed tailor-made ubiquitin protein ligases for substrate-selective extension of polyubiquitin chains with defined linkages. Application to the replication factor PCNA shows that alternative linkages confer distinct fates to the modified target. Proof-of-principle experiments demonstrate that the method of linkage reprogramming is generalizable to other monoubiquitylated proteins.
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3′-5′ exoribonuclease complex, suggesting a function in RNA ...metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN’s ability to activate cell growth-related genes but is required for MYCN’s ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
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•The MYCN oncoprotein exists in mutually exclusive DNA- and RNA-bound states•The conserved MYCBoxI mediates MYCN RNA binding in vitro and in cells•MYCN facilitates RNA processing as part of the nuclear exosome targeting complex•MYCN RNA binding supports S phase progression and tumor cell stress resilience
Papadopoulos et al. discover that MYCN, a chromatin-associated oncoprotein, engages RNA in vitro and in cells. DNA- and RNA-bound MYCN are mutually exclusive states with distinct interactomes and functions. The latter state facilitates RNA processing and prevents replication stress, thereby exposing a targetable vulnerability of aggressive MYCN-driven tumors.
The paralogs CUL4A and CUL4B assemble cullin‐RING E3 ubiquitin ligase (CRL) complexes regulating multiple chromatin‐associated cellular functions. Although they are structurally similar, we found ...that the unique N‐terminal extension of CUL4B is heavily phosphorylated during mitosis, and the phosphorylation pattern is perturbed in the CUL4B‐P50L mutation causing X‐linked intellectual disability (XLID). Phenotypic characterization and mutational analysis revealed that CUL4B phosphorylation is required for efficient progression through mitosis, controlling spindle positioning and cortical tension. While CUL4B phosphorylation triggers chromatin exclusion, it promotes binding to actin regulators and to two previously unrecognized CUL4B‐specific substrate receptors (DCAFs), LIS1 and WDR1. Indeed, co‐immunoprecipitation experiments and biochemical analysis revealed that LIS1 and WDR1 interact with DDB1, and their binding is enhanced by the phosphorylated N‐terminal domain of CUL4B. Finally, a human forebrain organoid model demonstrated that CUL4B is required to develop stable ventricular structures that correlate with onset of forebrain differentiation. Together, our study uncovers previously unrecognized DCAFs relevant for mitosis and brain development that specifically bind CUL4B, but not the CUL4B‐P50L patient mutant, by a phosphorylation‐dependent mechanism.
Synopsis
The cullin‐RING E3 ligase family CRL4 is based on two structurally similar scaffold subunits, CUL4A and CUL4B. Here, the unique N‐terminal extension in the CUL4B paralogue is found to be heavily phosphorylated in mitosis, which promotes the recruitment of a specific set of substrate receptors (DCAFs).
The N-terminus of CUL4B is phosphorylated during mitosis, and efficient phosphorylation is required for proper progression through mitosis.
CRL4B, but not CRL4A, recruits phospho‐specific DCAFs such as LIS1 and WDR1, which are involved in cytoskeleton regulation.
CUL4B is required to develop stable ventricular structures in human forebrain organoids.
A CUL4B patient mutation (P50L) alters mitotic phosphorylation and leads to X‐linked intellectual disability (XLID).
Heavy phosphorylation of a unique N‐terminal extension distinguishing CUL4B from its paralogue CUL4A promotes the recruitment of a specific set of substrate receptors involved in cytoskeleton regulation.
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been ...attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
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