PAXX and its paralogues synergistically direct DNA polymerase λ activity in DNA repair

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5′ end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.This work was supported by the Medical Research Council (MRC) UK

NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome

The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction.

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1

eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR

BackgroundRegulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood.ResultsHere, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5′UTR of target mRNAs directly upstream of the AUG start codon.ConclusionsOur data support a model whereby purine motifs towards the 3′ end of the 5′UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding.</p

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory

eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5'UTR

Background: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Results: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5′UTR of target mRNAs directly upstream of the AUG start codon. Conclusions: Our data support a model whereby purine motifs towards the 3′ end of the 5′UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding

Full-length NF-κB repressing factor contains an XRN2 binding domain.

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5'-3' exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif

DEAD-box helicase eIF4A2 inhibits CNOT7 deadenylation activity

The CCR4–NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4–NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4–NOT complex but instead inhibits CNOT7 activity. We identified a CCR4–NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4–NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4–NOT complex which results in different pathways for translational repression and mRNA deadenylation