Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated ...nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNA modifications have emerged as central regulators of gene expression programs. Amongst RNA modifications are N6-methyladenosine (m
A) and RNA 5-hydroxymethylcytosine (5hmC). While m
A is ...established as a versatile regulator of RNA metabolism, the functions of RNA 5hmC are unclear. Despite some evidence linking RNA modifications to immunity, their implications in gene expression control in macrophage development and functions remain unclear. Here we present a multi-omics dataset capturing different layers of the gene expression programs driving macrophage differentiation and polarisation. We obtained mRNA-Seq, m
A-IP-Seq, 5hmC-IP-Seq, Polyribo-Seq and LC-MS/MS data from monocytes and resting-, pro- and anti-inflammatory-like macrophages. We present technical validation showing high quality and correlation between samples for all datasets, and evidence of biological consistency of modelled macrophages at the transcriptomic, epitranscriptomic, translational and proteomic levels. This multi-omics dataset provides a resource for the study of RNA m
A and 5hmC in the context of macrophage biology and spans the gene expression process from transcripts to proteins.
Abstract
Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these ...cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.
The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more ...severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed “mouse kidney parvovirus” (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
Display omitted
•Inclusion body nephropathy is caused by mouse kidney parvovirus (MKPV)•MKPV is widely distributed in animal facilities in Australia and North America•MKPV is highly divergent from other mouse parvoviruses•Uncontrolled MKPV infection in immunocompromised mice results in renal failure
A kidney parvovirus found in multiple laboratory mouse colonies causes spontaneous nephropathy and represents a new tool for studying chronic kidney disease.
RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in ...monocyte-to-macrophage differentiation and polarisation is unclear. While m
6
A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m
6
A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m
6
A and 5 hmC reveals enrichment of m
6
A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m
6
A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m
6
A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m
6
A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.
Virilizer-like m
6
A methyltransferase-associated protein (VIRMA) maintains the stability of the m
6
A writer complex. Although VIRMA is critical for RNA m
6
A deposition, the impact of aberrant ...VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15–20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m
6
A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m
6
A-modified long non-coding RNA,
NEAT1
, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m
6
A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.
Chronic kidney disease (CKD) is a common cause of morbidity and mortality in domestic cats, but the cause is still largely elusive. While some viruses have been associated with this disease, none ...have been definitively implicated as causative. Recently, Rodent chaphamaparvovirus 1 was recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. A novel virus belonging to the same genus, Carnivore chaphamaparvovirus 2, was recently identified in the feces of cats with diarrhea. The goal of this study was to investigate the possible role of chaphamaparvoviruses including members of Rodent chaphamaparvovirus 1 and Carnivore chaphamaparvovirus 2 in the development of feline CKD. The presence of these viruses was retrospectively investigated in formalin-fixed paraffin-embedded feline kidney samples using polymerase chain reaction, in situ hybridization, and immunohistochemistry. Cats were divided into 3 groups: normal (N = 24), CKD (N = 26), and immunocompromised (N = 25). None of the kidney tissues from any of the 75 cats revealed the presence of chaphamaparvovirus DNA, RNA, or antigen. We conclude that viruses belonging to the chaphamaparvovirus genus are unlikely to contribute to the occurrence of feline CKD.
Fibroblast activation protein-α (FAP) can hydrolyze the post-proline bond. We identified endogenous substrates of FAP in fibroblasts that were previously naive to both FAP and its proteolytic ...activity. FAP-dependent cleavage sites were identified in many extracellular matrix (ECM) and ECM-associated proteins including collagens and lysyl oxidase-like-1, and CSF-1, CXCL-5 and C1qT6. Quantitative proteomic analysis implicated FAP in ECM-cell interactions, coagulation and metabolism. This study greatly expands the repertoire of FAP substrates and shows that FAP has a role in coagulation in the mouse.
Display omitted
Highlights
•Natural substrates of FAP were identified using degradomic and proteomic techniques and FAP gene knockout mouse derived embryonic fibroblasts stably transduced with enzymatically active or inactive FAP.•Terminal amine isotopic labelling of substrates (TAILS) based degradomics identified cleavage sites in collagens, and many other extracellular matrix (ECM) and associated proteins.•Cleavages of lysyl oxidase-like-1, CXCL-5, CSF-1 and C1qT6 by FAP were confirmed in vitro.•Differential metabolic labelling coupled with quantitative proteomic analysis implicated FAP in regulating proteins that are associated with ECM, ECM-cell interactions, coagulation, metabolism and wound healing.
Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.
Conventional dendritic cells (cDCs) are continuously replenished by bone marrow‐derived precursors called pre‐DCs, which traffic through the blood to peripheral tissues. Pre‐DCs are a heterogeneous ...population that includes cDC subset‐committed progenitors, namely pre‐cDC1 and pre‐cDC2, which give rise to mature cDC1 and cDC2, respectively. Regulation of pre‐DC subset trafficking is thought to aid the host response to immune challenge. However, the molecular cues regulating pre‐cDC1 versus pre‐cDC2 trafficking toward peripheral sites during homeostasis and disease remain elusive. Here, we report that pre‐cDC1 but not pre‐cDC2 express the T helper type 1‐associated chemokine receptor CXCR3. Moreover, we identify a cell‐intrinsic role for CXCR3 in the trafficking of pre‐cDC1 to melanoma tumors but not to non‐inflamed organs. We also show that tumor cDC1 numbers can be increased pharmacologically by targeting dipeptidyl peptidase‐4 (CD26), a negative regulator of CXCR3 ligands. Our findings demonstrate that pre‐cDC1 trafficking is regulated distinctly from pre‐cDC2, which is relevant for our understanding of the DC lineage in the context of cancer and inflammation.
The migration of conventional dendritic cell (cDC) precursors (pre‐DCs) is required for anti‐cancer immunity. The two subsets of cDC, cDC1 and cDC2, derive from committed progenitors called pre‐cDC1 and pre‐cDC2, respectively. In this study, we describe the differential expression of chemokine receptors by pre‐DC subsets and show that pre‐cDC1 but not pre‐cDC2 use CXCR3 to traffic into melanoma tumors in mice. We further show that the number of cDC1 within tumors can be increased through pharmacological inhibition of dipeptidyl‐peptidase 4 (DPP4), which normally inactivates the CXCR3 ligands CXCL9 and CXCL10.
PDF
