The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csb(m/m) developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders

DNA Damage: From Chronic Inflammation to Age-Related Deterioration

To lessen the wear and tear of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multi-cellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the micro-environment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression

DNA Damage Response and Metabolic Reprogramming in Health and Disease

Nuclear DNA damage contributes to cellular malfunction and the premature onset of age-related diseases, including cancer. Until recently, the canonical DNA damage response (DDR) was thought to represent a collection of nuclear processes that detect, signal and repair damaged DNA. However, recent evidence suggests that beyond nuclear events, the DDR rewires an intricate network of metabolic circuits, fine-tunes protein synthesis, trafficking, and secretion as well as balances growth with defense strategies in response to genotoxic insults. In this review, we discuss how the active DDR signaling mobilizes extranuclear and systemic responses to promote cellular homeostasis and organismal survival in health and disease

<i>s2p(-)</i> parasites show delayed transmission and growth, resulting in reduced virulence.

<p>(A) Kaplan-Meier analysis of time to patency after inoculation of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites into C57BL/6 mice (WT n = 9, <i>s2p(-)</i> n = 13). **, <i>P</i> < 0.01 (Log rank [Mantel-Cox] test). (B) Blood stage growth curve of the same mice showing the difference in growth rate of WT <i>vs</i>. <i>s2p(-)</i> parasites, alongside blood stage development of mice infected through bite back from infected <i>A</i>. <i>stephensi</i> mosquitoes (Wt bb <i>vs</i>. <i>s2p(-)</i> bb). In all cases mean values (± SD) are shown. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). Significance is shown as follows: Black stars: Sporozoite injection experiment, Grey stars: Bite back experiment. (C) Kaplan-Meier curve of mice developing experimental cerebral malaria (ECM) over time after injection of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites (WT n = 9, <i>s2p(-)</i> n = 13). ***, <i>P</i> < 0.001 (Log rank [Mantel-Cox] test). (D, E) Parasitaemia levels of C57BL/6 mice after infection with (D) 10,000 (<i>n</i> = 3 each) or (E) 1,000 (<i>n</i> = 5 each) WT or <i>s2p(-)</i> iRBCs, respectively, as determined by Giemsa stained blood smears. <i>s2p(-)</i> parasites exhibit slower growth rates compared to the WT line. Mean values (± SD) are shown *<i>P</i> < 0.05 **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). (F) Kaplan-Meier curve of time to ECM development after patency. A one day delay in ECM symptoms was observed in mice infected with 10,000 <i>s2p(-)</i> iRBCs (<i>P</i> = 0.11 (Log rank [Mantel-Cox] test). Mice infected with 1,000 WT iRBC developed ECM symptoms at day 8 (4/5) and 9 (1/5), while 2/5 <i>s2p(-)</i> infected mice developed ECM at day 11 and 3/5 remained free of ECM symptoms. **<i>P</i> < 0.01 (Log rank [Mantel-Cox] test).</p

<i>s2p(-)</i> parasites show no defects in sexual development nor sporogony.

<p>(A) Exflagellation assay showing formation of exflagellation centres. Mean values (±SD) from three independent experiments are shown. Differences were non-significant (Mann-Whitney test). (B) Ookinete conversion rates of WT and <i>s2p(-)</i> parasites after staining with an antibody against the surface antigen P28 and enumeration of ookinetes, zygotes and macrogametes. Shown are mean values (±SD) from three independent experiments. Differences were non-significant (2way-ANOVA). (C) Immunofluorescence analysis of <i>Anopheles gambiae</i> epithelia sheets infected with WT or <i>s2p(-)</i> parasites. Both strains induce an epithelial response as shown by the SRPN6 antibody (red). Ookinetes are stained with an antibody against surface protein P28 (green) and nuclei are stained with TO-PRO 3 (blue). Scale bar 10 μM. (D) Oocyst numbers of <i>s2p(-)</i> strain compared to the parental WT line after standard membrane feeding assay of <i>An</i>. <i>gambiae</i> mosquitoes from two independent experiments. Black bars show mean values (±SEM). Differences were non-significant (Mann-Whitney test).</p

Expression and localisation of <i>Pb</i>S2P.

<p>(A) Relative expression levels of <i>PbS2P</i> as determined by qRT-PCR from cDNAs of schizonts (schz), sporozoites (spz), 24h liver stages (LS24) and 48h liver stages (LS48). Transcript levels were normalised to <i>PbHSP70</i> and <i>GFP</i>. (B) Western blot analysis of <i>Pb</i>S2P-HA whole protein extract from purified schizonts of transgenic <i>PbS2P-HA</i> parasites using an α-HA antibody. <i>Pb</i>S2P-HA migrates at 35kDa. (C) Immunofluorescence analysis (IFA) of <i>Pb</i>S2P-HA merozoites, ookinete, oocyst, and salivary gland sporozoites using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and Hoechst stain for the nucleus (blue). For delineation of parasites the following antibodies (green) were used: α-HSP70, schizonts/merozoites; α-MTIP, ookinete and sporozoite; α-PbCap380, oocyst. Prominent localisation of <i>Pb</i>S2P in proximity to the nucleus is present in all invasive stages. Star, apical end of ookinete. Scale bar 5 μM.</p

<i>Pb</i>S2P shows partial co-localisation with the cis-Golgi marker ERD2.

<p>(A) Double labelling IFA of <i>P</i>. <i>berghei</i> schizont cultures using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and α-ERD2 as a Golgi marker (green) showing partial, or in some cases complete, co-localisation. Nuclei are stained with Hoechst (blue). Scale bar 5 μM.</p

<i>Plasmodium</i> M50 proteases.

<p>(A) Conserved catalytic motifs (HExxH and NxxPxxxxDG- highlighted red in grey boxes) from a multiple sequence alignment of S2P orthologues from <i>Plasmodium</i> species and related apicomplexan parasites. (B) 3D homology model of <i>Pb</i>S2P (right panel—PbANKA_1404100) using the open conformation of <i>Methanocaldococcus jannaschii</i> S2P (left panel—PDB id: 3B4R) as a template and Phyre2 as program. The first transmembrane domain is labelled in orange, the second to fourth in blue, and the fifth and sixth in lime green, respectively. The catalytic zinc atom is depicted in red and the catalytic residues are shown surrounding the zinc atom as blue sticks. The orientation within the lipid membrane is also indicated. (C) Magnification of the active site in the <i>Pb</i>S2P homology model, illustrating the structural conservation of the catalytic residues. Strictly conserved residues are shown as sticks and are labelled in black for <i>Pb</i>S2P and blue for the respective homologous amino acid residues in <i>M</i>. <i>jannaschii</i> S2P.</p

Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1(F/-)) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1(F/-) animal sera and are secreted in macrophage media after DNA damage. The Er1(F/-) EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging