Inflammasome protein scaffolds the DNA damage complex during tumor development.

Inflammasome sensors activate cellular signaling machineries to drive inflammation and cell death processes. Inflammasomes also control the development of certain diseases independently of canonical functions. Here, we show that the inflammasome protein NLR family CARD domain-containing protein 4 (NLRC4) attenuated the development of tumors in the Apcmin/+ mouse model. This response was independent of inflammasome signaling by NLRP3, NLRP6, NLR family apoptosis inhibitory proteins, absent in melanoma 2, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1 and caspase-11. NLRC4 interacted with the DNA-damage-sensing ataxia telangiectasia and Rad3-related (ATR)-ATR-interacting protein (ATRIP)-Ewing tumor-associated antigen 1 (ETAA1) complex to promote the recruitment of the checkpoint adapter protein claspin, licensing the activation of the kinase checkpoint kinase-1 (CHK1). Genotoxicity-induced activation of the NLRC4-ATR-ATRIP-ETAA1 complex drove the tumor-suppressing DNA damage response and CHK1 activation, and further attenuated the accumulation of DNA damage. These findings demonstrate a noninflammatory function of an inflammasome protein in promoting the DNA damage response and mediating protection against cancer.

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

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.

Orchestrated intron retention regulates normal granulocyte differentiation.

Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.

Variable calling of m6A and associated features in databases: a guide for end-users.

N6-methyladenosine (m$^{6}$A) is a widely-studied methylation to messenger RNAs, which has been linked to diverse cellular processes and human diseases. Numerous databases that collate m$^{6}$A profiles of distinct cell types have been created to facilitate quick and easy mining of m$^{6}$A signatures associated with cell-specific phenotypes. However, these databases contain inherent complexities that have not been explicitly reported, which may lead to inaccurate identification and interpretation of m$^{6}$A-associated biology by end-users who are unaware of them. Here, we review various m$^{6}$A-related databases, and highlight several critical matters. In particular, differences in peak-calling pipelines across databases drive substantial variability in both peak number and coordinates with only moderate reproducibility, and the inclusion of peak calls from early m$^{6}$A sequencing protocols may lead to the reporting of false positives or negatives. The awareness of these matters will help end-users avoid the inclusion of potentially unreliable data in their studies and better utilize m$^{6}$A databases to derive biologically meaningful results.

Intron retention: importance, challenges, and opportunities.

Recent landmark discoveries have underpinned the physiological importance of intron retention (IR) across multiple domains of life and revealed an unexpected breath of functions in a large variety of biological processes. Despite significant progress in the field, some challenges remain. Once solved, opportunities will arise for discovering more functions of IR.

The multifaceted effects of YTHDC1-mediated nuclear m6A recognition.

N6-methyladenosine or m6A modification to mRNAs is now recognised as a key regulator of gene expression and protein translation. The fate of m6A-modified mRNAs is decoded by m6A readers, mostly found in the cytoplasm, except for the nuclear-localised YTHDC1. While earlier studies have implicated YTHDC1-m6A functions in alternative splicing and mRNA export, recent literature has expanded its close association to the chromatin-associated, noncoding and regulatory RNAs to fine-tune transcription and gene expression in cells. Here, we summarise current progress in the study of YTHDC1 function in cells, highlighting its multiple modes of action in regulating gene expression, and propose the formation of YTHDC1 nuclear condensates as a general mechanism that underlies its diverse functions in the nucleus.

SpliceWiz: interactive analysis and visualization of alternative splicing in R.

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz, an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz).

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

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 m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A 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 m6A 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 m6A. 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.

The m6A-epitranscriptome in brain plasticity, learning and memory.

Activity-dependent gene expression and protein translation underlie the ability of neurons to dynamically adjust their synaptic strength in response to sensory experience and during learning. The emerging field of epitranscriptomics (RNA modifications) has rapidly shifted our views on the mechanisms that regulate gene expression. Among hundreds of biochemical modifications on RNA, N6-methyladenosine (m6A) is the most abundant reversible mRNA modification in the brain. Its dynamic nature and ability to regulate all aspects of mRNA processing have positioned m6A as an important and versatile regulator of nervous system functions, including neuronal plasticity, learning and memory. In this review, we summarise recent experimental evidence that supports the role of m6A signalling in learning and memory, as well as providing an overview of the underlying molecular mechanisms in neurons. We also discuss the consequences of perturbed m6A signalling and/or its regulatory networks which are increasingly being linked to various cognitive disorders in humans.

CYP eicosanoid pathway mediates colon cancer-promoting effects of dietary linoleic acid.

Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω-6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer-promoting effects. Using LC-MS/MS-based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer-promoting effects of LA, since the LA-rich diet fails to exacerbate colon cancer in CYP monooxygenase-deficient mice. Finally, CYP monooxygenase mediates the pro-cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota-dependent mechanisms. Overall, these results support that CYP monooxygenase-mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects.

Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A 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 m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A 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.

MLH1-methylated endometrial cancer under 60 years of age as the "sentinel" cancer in female carriers of high-risk constitutional MLH1 epimutation.

OBJECTIVE: Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to determine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1-methylated tumors. METHODS: We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4, <60 years), and (ii) two population-based cohorts; "Columbus-area" (n = 68, all ages) and "Ohio Colorectal Cancer Prevention Initiative (OCCPI)" (n = 24, <60 years). RESULTS: Constitutional MLH1 methylation was identified in three out of four patients diagnosed between 36 and 59 years from cancer clinics. Two had mono-/hemiallelic epimutation (∼50% alleles methylated). One with multiple primaries had low-level mosaicism in normal tissues and somatic "second-hits" affecting the unmethylated allele in all tumors, demonstrating causation. In the population-based cohorts, all 68 cases from the Columbus-area cohort were negative and low-level mosaic constitutional MLH1 methylation was identified in one patient aged 36 years out of 24 from the OCCPI cohort, representing one of six (∼17%) patients <50 years and one of 45 patients (∼2%) <60 years in the combined cohorts. EC was the first/dual-first cancer in three patients with underlying constitutional MLH1 methylation. CONCLUSIONS: A correct diagnosis at first presentation of cancer is important as it will significantly alter clinical management. Screening for constitutional MLH1 methylation is warranted in patients with early-onset EC or synchronous/metachronous tumors (any age) displaying MLH1 methylation.

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

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.

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

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.

CCM2L (Cerebral Cavernous Malformation 2 Like) Deletion Aggravates Cerebral Cavernous Malformation Through Map3k3-KLF Signaling Pathway.

[Figure: see text].

Ctcf haploinsufficiency mediates intron retention in a tissue-specific manner.

CTCF is a master regulator of gene transcription and chromatin organisation with occupancy at thousands of DNA target sites genome-wide. While CTCF is essential for cell survival, CTCF haploinsufficiency is associated with tumour development and hypermethylation. Increasing evidence demonstrates CTCF as a key player in several mechanisms regulating alternative splicing (AS), however, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in five tissues from Ctcf hemizygous (Ctcf+/-) mice. Reduced Ctcf levels caused distinct tissue-specific differences in gene expression and AS in all tissues. An increase in intron retention (IR) was observed in Ctcf+/- liver and kidney. In liver, this specifically impacted genes associated with cytoskeletal organisation, splicing and metabolism. Strikingly, most differentially retained introns were short, with a high GC content and enriched in Ctcf binding sites in their proximal upstream genomic region. This study provides new insights into the effects of CTCF haploinsufficiency on organ transcriptomes and the role of CTCF in AS regulation.

Challenges in defining the role of intron retention in normal biology and disease.

RNA sequencing has revealed a striking diversity in transcriptomic complexity, to which alternative splicing is a major contributor. Intron retention (IR) is a conserved form of alternative splicing that was originally overlooked in normal mammalian physiology and development, due mostly to difficulties in its detection. IR has recently been revealed as an independent mechanism of controlling and enhancing the complexity of gene expression. IR facilitates rapid responses to biological stimuli, is involved in disease pathogenesis, and can generate novel protein isoforms. Many challenges, however, remain in detecting and quantifying retained introns and in determining their effects on cellular phenotype. In this review, we provide an overview of these challenges, and highlight approaches that can be used to address them.

Differential chemokine receptor expression and usage by pre-cDC1 and pre-cDC2.

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.

A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages.

Intron retention (IR) is a form of alternative splicing that can impact mRNA levels through nonsense-mediated decay or by nuclear mRNA detention. A complex, dynamic IR pattern has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We globally assessed IR in primary maturing mammalian erythroid and megakaryocyte (MK) lineages as well as their common progenitor cells (MEPs). Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes with an overwhelming loss of IR in erythroid cells and MKs compared to MEPs. Moreover, complex IR patterns were seen throughout murine erythroid maturation. Similarly complex patterns were observed in human erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and MKs, and overlapping gene expression patterns, the MK IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. Importantly, our results suggest that IR serves to broadly regulate mRNA levels. These findings highlight the importance of this understudied form of alternative splicing in gene regulation and provide a useful resource for studies on gene expression in the MK and erythroid lineages.