Paternal starvation affects metabolic gene expression during zebrafish offspring development and lifelong fitness.

Dietary restriction in the form of fasting is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of long-term fasting and starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of fasting/starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected increased larval growth rate in F1 offspring from starved males and more malformed embryos at 24 h post-fertilisation in F2 offspring produced by F1 offspring from starved males. Comparing the transcriptomes of F1 embryos sired by starved and fed fathers revealed robust and reproducible increased expression of muscle composition genes but lower expression of lipid metabolism and lysosome genes in embryos from starved fathers. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal effects on extra-embryonic tissues which are loaded with maternal factors. We compared the embryo transcriptomes to published adult transcriptome datasets and found comparable repressive effects of starvation on metabolism-associated genes. These similarities suggest a physiologically relevant, directed and potentially adaptive response transmitted by the father, independently from the offspring's nutritional state, which was defined by the mother.

LINE-1 regulates cortical development by acting as long non-coding RNAs.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

In silico characterisation of minor wave genes and LINE-1s transcriptional dynamics at murine zygotic genome activation.

Introduction: In mouse, the zygotic genome activation (ZGA) is coordinated by MERVL elements, a class of LTR retrotransposons. In addition to MERVL, another class of retrotransposons, LINE-1 elements, recently came under the spotlight as key regulators of murine ZGA. In particular, LINE-1 transcripts seem to be required to switch-off the transcriptional program started by MERVL sequences, suggesting an antagonistic interplay between LINE-1 and MERVL pathways. Methods: To better investigate the activities of LINE-1 and MERVL elements at ZGA, we integrated publicly available transcriptomics (RNA-seq), chromatin accessibility (ATAC-seq) and Pol-II binding (Stacc-seq) datasets and characterised the transcriptional and epigenetic dynamics of such elements during murine ZGA. Results: We identified two likely distinct transcriptional activities characterising the murine zygotic genome at ZGA onset. On the one hand, our results confirmed that ZGA minor wave genes are preferentially transcribed from MERVL-rich and gene-dense genomic compartments, such as gene clusters. On the other hand, we identified a set of evolutionary young and likely transcriptionally autonomous LINE-1s located in intergenic and gene-poor regions showing, at the same stage, features such as open chromatin and RNA Pol II binding suggesting them to be, at least, poised for transcription. Discussion: These results suggest that, across evolution, transcription of two different classes of transposable elements, MERVLs and LINE-1s, have likely been confined in genic and intergenic regions respectively in order to maintain and regulate two successive transcriptional programs at ZGA.

Exploratory analysis of L1 retrotransposons expression in autism.

BACKGROUND: Autism spectrum disorder (ASD) is a set of highly heterogeneous neurodevelopmental diseases whose genetic etiology is not completely understood. Several investigations have relied on transcriptome analysis from peripheral tissues to dissect ASD into homogenous molecular phenotypes. Recently, analysis of changes in gene expression from postmortem brain tissues has identified sets of genes that are involved in pathways previously associated with ASD etiology. In addition to protein-coding transcripts, the human transcriptome is composed by a large set of non-coding RNAs and transposable elements (TEs). Advancements in sequencing technologies have proven that TEs can be transcribed in a regulated fashion, and their dysregulation might have a role in brain diseases. METHODS: We exploited published datasets comprising RNA-seq data from (1) postmortem brain of ASD subjects, (2) in vitro cell cultures where ten different ASD-relevant genes were knocked out and (3) blood of discordant siblings. We measured the expression levels of evolutionarily young full-length transposable L1 elements and characterized the genomic location of deregulated L1s assessing their potential impact on the transcription of ASD-relevant genes. We analyzed every sample independently, avoiding to pool together the disease subjects to unmask the heterogeneity of the molecular phenotypes. RESULTS: We detected a strong upregulation of intronic full-length L1s in a subset of postmortem brain samples and in in vitro differentiated neurons from iPSC knocked out for ATRX. L1 upregulation correlated with an high number of deregulated genes and retained introns. In the anterior cingulate cortex of one subject, a small number of significantly upregulated L1s overlapped with ASD-relevant genes that were significantly downregulated, suggesting the possible existence of a negative effect of L1 transcription on host transcripts. LIMITATIONS: Our analyses must be considered exploratory and will need to be validated in bigger cohorts. The main limitation is given by the small sample size and by the lack of replicates for postmortem brain samples. Measuring the transcription of locus-specific TEs is complicated by the repetitive nature of their sequence, which reduces the accuracy in mapping sequencing reads to the correct genomic locus. CONCLUSIONS: L1 upregulation in ASD appears to be limited to a subset of subjects that are also characterized by a general deregulation of the expression of canonical genes and an increase in intron retention. In some samples from the anterior cingulate cortex, L1s upregulation seems to directly impair the expression of some ASD-relevant genes by a still unknown mechanism. L1s upregulation may therefore identify a group of ASD subjects with common molecular features and helps stratifying individuals for novel strategies of therapeutic intervention.

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides.

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.

The miR-430 locus with extreme promoter density forms a transcription body during the minor wave of zygotic genome activation.

In anamniote embryos, the major wave of zygotic genome activation starts during the mid-blastula transition. However, some genes escape global genome repression, are activated substantially earlier, and contribute to the minor wave of genome activation. The mechanisms underlying the minor wave of genome activation are little understood. We explored the genomic organization and cis-regulatory mechanisms of a transcription body, in which the minor wave of genome activation is first detected in zebrafish. We identified the miR-430 cluster as having excessive copy number and the highest density of Pol-II-transcribed promoters in the genome, and this is required for forming the transcription body. However, this transcription body is not essential for, nor does it encompasse, minor wave transcription globally. Instead, distinct minor-wave-specific promoter architecture suggests that promoter-autonomous mechanisms regulate the minor wave of genome activation. The minor-wave-specific features also suggest distinct transcription initiation mechanisms between the minor and major waves of genome activation.

Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

Transposons Acting as Competitive Endogenous RNAs: In-Silico Evidence from Datasets Characterised by L1 Overexpression.

LINE L1 are transposable elements that can replicate within the genome by passing through RNA intermediates. The vast majority of these element copies in the human genome are inactive and just between 100 and 150 copies are still able to mobilize. During evolution, they could have been positively selected for beneficial cellular functions. Nonetheless, L1 deregulation can be detrimental to the cell, causing diseases such as cancer. The activity of miRNAs represents a fundamental mechanism for controlling transcript levels in somatic cells. These are a class of small non-coding RNAs that cause degradation or translational inhibition of their target transcripts. Beyond this, competitive endogenous RNAs (ceRNAs), mostly made by circular and non-coding RNAs, have been seen to compete for the binding of the same set of miRNAs targeting protein coding genes. In this study, we have investigated whether autonomously transcribed L1s may act as ceRNAs by analyzing public dataset in-silico. We observed that genes sharing miRNA target sites with L1 have a tendency to be upregulated when L1 are overexpressed, suggesting the possibility that L1 might act as ceRNAs. This finding will help in the interpretation of transcriptomic responses in contexts characterized by the specific activation of transposons.

TEspeX: consensus-specific quantification of transposable element expression preventing biases from exonized fragments.

SUMMARY: Transposable elements (TEs) play key roles in crucial biological pathways. Therefore, several tools enabling the quantification of their expression were recently developed. However, many of the existing tools lack the capability to distinguish between the transcription of autonomously expressed TEs and TE fragments embedded in canonical coding/non-coding non-TE transcripts. Consequently, an apparent change in the expression of a given TE may simply reflect the variation in the expression of the transcripts containing TE-derived sequences. To overcome this issue, we have developed TEspeX, a pipeline for the quantification of TE expression at the consensus level. TEspeX uses Illumina RNA-seq short reads to quantify TE expression avoiding counting reads deriving from inactive TE fragments embedded in canonical transcripts. AVAILABILITY AND IMPLEMENTATION: The tool is implemented in python3, distributed under the GNU General Public License (GPL) and available on Github at https://github.com/fansalon/TEspeX (Zenodo URL: https://doi.org/10.5281/zenodo.6800331). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Meta-Analysis Suggests That Intron Retention Can Affect Quantification of Transposable Elements from RNA-Seq Data.

Transposable elements (TEs), also known as "jumping genes", are repetitive sequences with the capability of changing their location within the genome. They are key players in many different biological processes in health and disease. Therefore, a reliable quantification of their expression as transcriptional units is crucial to distinguish between their independent expression and the transcription of their sequences as part of canonical transcripts. TEs quantification faces difficulties of different types, the most important one being low reads mappability due to their repetitive nature preventing an unambiguous mapping of reads originating from their sequences. A large fraction of TEs fragments localizes within introns, which led to the hypothesis that intron retention (IR) can be an additional source of bias, potentially affecting accurate TEs quantification. IR occurs when introns, normally removed from the mature transcript by the splicing machinery, are maintained in mature transcripts. IR is a widespread mechanism affecting many different genes with cell type-specific patterns. We hypothesized that, in an RNA-seq experiment, reads derived from retained introns can introduce a bias in the detection of overlapping, independent TEs RNA expression. In this study we performed meta-analysis using public RNA-seq data from lymphoblastoid cell lines and show that IR can impact TEs quantification using established tools with default parameters. Reads mapped on intronic TEs were indeed associated to the expression of TEs and influence their correct quantification as independent transcriptional units. We confirmed these results using additional independent datasets, demonstrating that this bias does not appear in samples where IR is not present and that differential TEs expression does not impact on IR quantification. We concluded that IR causes the over-quantification of intronic TEs and differential IR might be confused with differential TEs expression. Our results should be taken into account for a correct quantification of TEs expression from RNA-seq data, especially in samples in which IR is abundant.

Supporting Cells of the Human Olfactory Epithelium Co-Express the Lipid Scramblase TMEM16F and ACE2 and May Cause Smell Loss by SARS-CoV-2 Spike-Induced Syncytia.

BACKGROUND/AIMS: Quantitative and qualitative alterations in the sense of smell are well established symptoms of COVID-19. Some reports have shown that non-neuronal supporting (also named sustentacular) cells of the human olfactory epithelium co-express ACE2 and TMPRSS2 necessary for SARS-CoV-2 infection. In COVID-19, syncytia were found in many tissues but were not investigated in the olfactory epithelium. Some studies have shown that syncytia in some tissues are formed when SARS-CoV-2 Spike expressed at the surface of an infected cell binds to ACE2 on another cell, followed by activation of the scramblase TMEM16F (also named ANO6) which exposes phosphatidylserine to the external side of the membrane. Furthermore, niclosamide, an approved antihelminthic drug, inhibits Spike-induced syncytia by blocking TMEM16F activity. The aim of this study was to investigate if proteins involved in Spike-induced syncytia formation, i.e., ACE2 and TMEM16F, are expressed in the human olfactory epithelium. METHODS: We analysed a publicly available single-cell RNA-seq dataset from human nasal epithelium and performed immunohistochemistry in human nasal tissues from biopsies. RESULTS: We found that ACE2 and TMEM16F are co-expressed both at RNA and protein levels in non-neuronal supporting cells of the human olfactory epithelium. CONCLUSION: Our results provide the first evidence that TMEM16F is expressed in human olfactory supporting cells and indicate that syncytia formation, that could be blocked by niclosamide, is one of the pathogenic mechanisms worth investigating in COVID-19 smell loss.

Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development.

The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).

Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain.

BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

Trade-off between sex and growth in diatoms: Molecular mechanisms and demographic implications.

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities.

Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. Here we show that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.

Natural SINEUP RNAs in Autism Spectrum Disorders: RAB11B-AS1 Dysregulation in a Neuronal CHD8 Suppression Model Leads to RAB11B Protein Increase.

CHD8 represents one of the highest confidence genetic risk factors implied in Autism Spectrum Disorders, with most mutations leading to CHD8 haploinsufficiency and the insurgence of specific phenotypes, such as macrocephaly, facial dysmorphisms, intellectual disability, and gastrointestinal complaints. While extensive studies have been conducted on the possible consequences of CHD8 suppression and protein coding RNAs dysregulation during neuronal development, the effects of transcriptional changes of long non-coding RNAs (lncRNAs) remain unclear. In this study, we focused on a peculiar class of natural antisense lncRNAs, SINEUPs, that enhance translation of a target mRNA through the activity of two RNA domains, an embedded transposable element sequence and an antisense region. By looking at dysregulated transcripts following CHD8 knock down (KD), we first identified RAB11B-AS1 as a potential SINEUP RNA for its domain configuration. Then we demonstrated that such lncRNA is able to increase endogenous RAB11B protein amounts without affecting its transcriptional levels. RAB11B has a pivotal role in vesicular trafficking, and mutations on this gene correlate with intellectual disability and microcephaly. Thus, our study discloses an additional layer of molecular regulation which is altered by CHD8 suppression. This represents the first experimental confirmation that naturally occurring SINEUP could be involved in ASD pathogenesis and underscores the importance of dysregulation of functional lncRNAs in neurodevelopment.

SINEUPs: a novel toolbox for RNA therapeutics.

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

The prolyl-isomerase PIN1 is essential for nuclear Lamin-B structure and function and protects heterochromatin under mechanical stress.

Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies.

Type I interferon activation in RAS-associated autoimmune leukoproliferative disease (RALD).

RAS-associated autoimmune leukoproliferative disease (RALD) is a rare immune dysregulation syndrome caused by somatic gain-of-function mutations of either NRAS or KRAS gene in hematopoietic cells. We describe a 27-year-old patient presenting at 5 months of age with recurrent infections and generalized lymphadenopathy who developed a complex multi-organ autoimmune syndrome with hypogammaglobulinemia, partially controlled with oral steroids, hydroxichloroquine, mofetil mycophenolate and IVIG prophylaxis. Activation of type I interferon pathway was observed in peripheral blood. Since 18 years of age, the patient developed regenerative nodular hyperplasia of the liver evolving into hepatopulmonary syndrome. Whole exome sequencing analysis of the peripheral blood DNA showed the NRAS p.Gly13Asp mutation validated as somatic. Our report highlights the possibility of detecting somatic NRAS gene mutations in patients with inflammatory immune dysregulation and type I interferon activation.

Prolyl 3-Hydroxylase 2 Is a Molecular Player of Angiogenesis.

Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3H2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.