The mRNA-bound proteome of Leishmania mexicana : novel genetic insight into an ancient parasite

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Utilizing optimised crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1,400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, while the crosslinked RBPome is magnitudes more enriched the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L.mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression

miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity, however its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ 38 T cells express high levels of miR-132 and that T cell activation leads to miR-132 upregulation. The transcriptomic hallmark of splenic CD4+ 40 T cells lacking the miR 132/212 cluster during chronic infection is an increase in mRNAs levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with Leishmania donovani miR-132-/- CD4+ T cells display enhanced expression of IL-10 and decreased IFNg. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in miR-132-/- Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4+ 50 T cell activation and protective immunity against pathogen

Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate -catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of - catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of -catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signallin

Maintenance of epigenetic landscape requires CIZ1 and is corrupted in differentiated fibroblasts in long-term culture

The inactive X chromosome (Xi) serves as a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes (PRC1/2). Here we show that Xi transiently relocates from the nuclear periphery towards the interior during its replication, in a process dependent on CIZ1. Compromised relocation of Xi in CIZ1-null primary mouse embryonic fibroblasts is accompanied by loss of PRC-mediated H2AK119Ub1 and H3K27me3, increased solubility of PRC2 catalytic subunit EZH2, and genome-wide deregulation of polycomb-regulated genes. Xi position in S phase is also corrupted in cells adapted to long-term culture (WT or CIZ1-null), and also accompanied by specific changes in EZH2 and its targets. The data are consistent with the idea that chromatin relocation during S phase contributes to maintenance of epigenetic landscape in primary cells, and that elevated soluble EZH2 is part of an error-prone mechanism by which modifying enzyme meets template when chromatin relocation is compromised

Essential roles for deubiquitination in Leishmania life cycle progression

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19

Epigenetic instability caused by absence of CIZ1 drives transformation during quiescence cycles

Abstract Background Cip1-interacting zinc finger protein 1 (CIZ1) forms RNA-dependent protein assemblies that stabilise epigenetic state, notable at the inactive X chromosome in females. CIZ1 has been linked with a range of human cancers and in mice genetic deletion of CIZ1 manifests as hyperproliferative lymphoid lineages in females. This suggests that its role in maintenance of epigenetic stability is linked with disease. Results Here, we show that male and female CIZ1-null primary murine fibroblasts have reduced H4K20me1 and that this compromises nuclear condensation on entry to quiescence. Global transcriptional repression remains intact in condensation-deficient CIZ1-null cells; however, a subset of genes linked with chromatin condensation and homology-directed DNA repair are perturbed. Failure to condense is phenotypically mimicked by manipulation of the H4K20me1 methyltransferase, SET8, in WT cells and partially reverted in CIZ1-null cells upon re-expression of CIZ1. Crucially, during exit from quiescence, nuclear decondensation remains active, so that repeated entry and exit cycles give rise to expanded nuclei susceptible to mechanical stress, DNA damage checkpoint activation, and downstream emergence of transformed proliferative colonies. Conclusions Our results demonstrate a role for CIZ1 in chromatin condensation on entry to quiescence and explore the consequences of this defect in CIZ1-null cells. Together, the data show that CIZ1’s protection of the epigenome guards against genome instability during quiescence cycles. This identifies loss of CIZ1 as a potentially devastating vulnerability in cells that undergo cycles of quiescence entry and exit