Alternative splicing induced by bacterial pore-forming toxins sharpens CIRBP-mediated cell response to Listeria infection.

Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based types of regulation, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of 'poison exons'. We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin or aerolysin) led to the dephosphorylation of SRSFs via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.

Increased plasma DYRK1A with aging may protect against neurodegenerative diseases.

Early markers are needed for more effective prevention of Alzheimer's disease. We previously showed that individuals with Alzheimer's disease have decreased plasma DYRK1A levels compared to controls. We assessed DYRK1A in the plasma of cognitively healthy elderly volunteers, individuals with either Alzheimer's disease (AD), tauopathies or Down syndrome (DS), and in lymphoblastoids from individuals with DS. DYRK1A levels were inversely correlated with brain amyloid ß burden in asymptomatic elderly individuals and AD patients. Low DYRK1A levels were also detected in patients with tauopathies. Individuals with DS had higher DYRK1A levels than controls, although levels were lower in individuals with DS and with dementia. These data suggest that plasma DYRK1A levels could be used for early detection of at risk individuals of AD and for early detection of AD. We hypothesize that lack of increase of DYRK1A at middle age (40-50 years) could be a warning before the cognitive decline, reflecting increased risk for AD.

A conserved molecular template underlies color pattern diversity in estrildid finches.

The color patterns that adorn animals' coats not only exhibit extensive diversity linked to various ecological functions but also display recurrences in geometry, orientation, or body location. How processes of pattern formation shape such phenotypic trends remains a mystery. Here, we surveyed plumage color patterns in passerine finches displaying extreme apparent variation and identified a conserved set of color domains. We linked these domains to putative embryonic skin regions instructed by early developmental tissues and outlined by the combinatory expression of few genetic markers. We found that this embryonic prepattern is largely conserved in birds displaying drastic color differences in the adult, interspecies variation resulting from the masking or display of each domain depending on their coloration. This work showed that a simple molecular landscape serves as common spatial template to extensive color pattern variation in finches, revealing that early conserved landmarks and molecular pathways are a major cause of phenotypic trends.

Expression Pattern of Purinergic Signaling Components in Colorectal Cancer Cells and Differential Cellular Outcomes Induced by Extracellular ATP and Adenosine.

The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through "RNA-Sequencing" (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca2+ observed after ATP exposure in all CRC cell lines, Ca2+ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.

Comparative transcriptome analysis at the onset of speciation in a mimetic butterfly-The Ithomiini Melinaea marsaeus.

Ecological speciation entails divergent selection on specific traits and ultimately on the developmental pathways responsible for these traits. Selection can act on gene sequences but also on regulatory regions responsible for gene expression. Mimetic butterflies are a relevant system for speciation studies because wing colour pattern (WCP) often diverges between closely related taxa and is thought to drive speciation through assortative mating and increased predation on hybrids. Here, we generate the first transcriptomic resources for a mimetic butterfly of the tribe Ithomiini, Melinaea marsaeus, to examine patterns of differential expression between two subspecies and between tissues that express traits that likely drive reproductive isolation; WCP and chemosensory genes. We sequenced whole transcriptomes of three life stages to cover a large catalogue of transcripts, and we investigated differential expression between subspecies in pupal wing discs and antennae. Eighteen known WCP genes were expressed in wing discs and 115 chemosensory genes were expressed in antennae, with a remarkable diversity of chemosensory protein genes. Many transcripts were differentially expressed between subspecies, including two WCP genes and one odorant receptor. Our results suggest that in M. marsaeus the same genes as in other mimetic butterflies are involved in traits causing reproductive isolation, and point at possible candidates for the differences in those traits between subspecies. Differential expression analyses of other developmental stages and body organs and functional studies are needed to confirm and expand these results. Our work provides key resources for comparative genomics in mimetic butterflies, and more generally in Lepidoptera.

Distinctive Cellular and Metabolic Reprogramming in Porcine Lung Mononuclear Phagocytes Infected With Type 1 PRRSV Strains.

Porcine reproductive and respiratory syndrome (PRRS) has an extensive impact on pig production. The causative virus (PRRSV) is divided into two species, PRRSV-1 (European origin) and PRRSV-2 (North American origin). Within PRRSV-1, PRRSV-1.3 strains, such as Lena, are more pathogenic than PRRSV-1.1 strains, such as Flanders 13 (FL13). To date, the molecular interactions of PRRSV with primary lung mononuclear phagocyte (MNP) subtypes, including conventional dendritic cells types 1 (cDC1) and 2 (cDC2), monocyte-derived DCs (moDC), and pulmonary intravascular macrophages (PIM), have not been thoroughly investigated. Here, we analyze the transcriptome profiles of in vivo FL13-infected parenchymal MNP subpopulations and of in vitro FL13- and Lena-infected parenchymal MNP. The cell-specific expression profiles of in vivo sorted cells correlated with their murine counterparts (AM, cDC1, cDC2, moDC) with the exception of PIM. Both in vivo and in vitro, FL13 infection altered the expression of a low number of host genes, and in vitro infection with Lena confirmed the higher ability of this strain to modulate host response. Machine learning (ML) and gene set enrichment analysis (GSEA) unraveled additional relevant genes and pathways modulated by FL13 infection that were not identified by conventional analyses. GSEA increased the cellular pathways enriched in the FL13 data set, but ML allowed a more complete comprehension of functional profiles during FL13 in vitro infection. Data indicates that cellular reprogramming differs upon Lena and FL13 infection and that the latter might keep antiviral and inflammatory macrophage/DC functions silent. Although the slow replication kinetics of FL13 likely contribute to differences in cellular gene expression, the data suggest distinct mechanisms of interaction of the two viruses with the innate immune system during early infection.

Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection.

BACKGROUND: Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells. RESULTS: ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build "splice trees", a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. CONCLUSION: ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.

Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B monoubiquitination.

BACKGROUND: The functional determinants of H3K4me3, their potential dependency on histone H2B monoubiquitination, and their contribution to defining transcriptional regimes are poorly defined in plant systems. Unlike in Saccharomyces cerevisiae, where a single SET1 protein catalyzes H3K4me3 as part of COMPlex of proteins ASsociated with Set1 (COMPASS), in Arabidopsis thaliana, this activity involves multiple histone methyltransferases. Among these, the plant-specific SET DOMAIN GROUP 2 (SDG2) has a prominent role. RESULTS: We report that SDG2 co-regulates hundreds of genes with SWD2-like b (S2Lb), a plant ortholog of the Swd2 axillary subunit of yeast COMPASS. We show that S2Lb co-purifies with the AtCOMPASS core subunit WDR5, and both S2Lb and SDG2 directly influence H3K4me3 enrichment over highly transcribed genes. S2Lb knockout triggers pleiotropic developmental phenotypes at the vegetative and reproductive stages, including reduced fertility and seed dormancy. However, s2lb seedlings display little transcriptomic defects as compared to the large repertoire of genes targeted by S2Lb, SDG2, or H3K4me3, suggesting that H3K4me3 enrichment is important for optimal gene induction during cellular transitions rather than for determining on/off transcriptional status. Moreover, unlike in budding yeast, most of the S2Lb and H3K4me3 genomic distribution does not rely on a trans-histone crosstalk with histone H2B monoubiquitination. CONCLUSIONS: Collectively, this study unveils that the evolutionarily conserved COMPASS-like complex has been co-opted by the plant-specific SDG2 histone methyltransferase and mediates H3K4me3 deposition through an H2B monoubiquitination-independent pathway in Arabidopsis.

Neuroglobin Can Prevent or Reverse Glaucomatous Progression in DBA/2J Mice.

Mitochondrial dysfunction is responsible for hereditary optic neuropathies. We wished to determine whether preserving mitochondrial bioenergetics could prevent optic neuropathy in a reliable model of glaucoma. DBA/2J mice exhibit elevated intraocular pressure, progressive degeneration of their retinal ganglion cells, and optic neuropathy that resembles glaucoma. We established that glaucoma in these mice is directly associated with mitochondrial dysfunction: respiratory chain activity was compromised in optic nerves 5 months before neuronal loss began, and the amounts of some mitochondrial proteins were reduced in retinas of glaucomatous mice. One of these proteins is neuroglobin, which has a neuroprotective function. Therefore, we investigated whether gene therapy aimed at restoring neuroglobin levels in the retina via ocular administration of an adeno-associated viral vector could reduce neuronal degeneration. The approach of treating 2-month-old mice impeded glaucoma development: few neurons died and respiratory chain activity and visual cortex activity were comparable to those in young, asymptomatic mice. When the treatment was performed in 8-month-old mice, the surviving neurons acquired new morphologic and functional properties, leading to the preservation of visual cortex activity and respiratory chain activity. The beneficial effects of neuroglobin in DBA/2J retinas confirm this protein to be a promising candidate for treating glaucoma.