IRFinder-S: a comprehensive suite to discover and explore intron retention.

Accurate quantification and detection of intron retention levels require specialized software. Building on our previous software, we create a suite of tools called IRFinder-S, to analyze and explore intron retention events in multiple samples. Specifically, IRFinder-S allows a better identification of true intron retention events using a convolutional neural network, allows the sharing of intron retention results between labs, integrates a dynamic database to explore and contrast available samples, and provides a tested method to detect differential levels of intron retention.

A cell-to-patient machine learning transfer approach uncovers novel basal-like breast cancer prognostic markers amongst alternative splice variants.

BACKGROUND: Breast cancer is amongst the 10 first causes of death in women worldwide. Around 20% of patients are misdiagnosed leading to early metastasis, resistance to treatment and relapse. Many clinical and gene expression profiles have been successfully used to classify breast tumours into 5 major types with different prognosis and sensitivity to specific treatments. Unfortunately, these profiles have failed to subclassify breast tumours into more subtypes to improve diagnostics and survival rate. Alternative splicing is emerging as a new source of highly specific biomarkers to classify tumours in different grades. Taking advantage of extensive public transcriptomics datasets in breast cancer cell lines (CCLE) and breast cancer tumours (TCGA), we have addressed the capacity of alternative splice variants to subclassify highly aggressive breast cancers. RESULTS: Transcriptomics analysis of alternative splicing events between luminal, basal A and basal B breast cancer cell lines identified a unique splicing signature for a subtype of tumours, the basal B, whose classification is not in use in the clinic yet. Basal B cell lines, in contrast with luminal and basal A, are highly metastatic and express epithelial-to-mesenchymal (EMT) markers, which are hallmarks of cell invasion and resistance to drugs. By developing a semi-supervised machine learning approach, we transferred the molecular knowledge gained from these cell lines into patients to subclassify basal-like triple negative tumours into basal A- and basal B-like categories. Changes in splicing of 25 alternative exons, intimately related to EMT and cell invasion such as ENAH, CD44 and CTNND1, were sufficient to identify the basal-like patients with the worst prognosis. Moreover, patients expressing this basal B-specific splicing signature also expressed newly identified biomarkers of metastasis-initiating cells, like CD36, supporting a more invasive phenotype for this basal B-like breast cancer subtype. CONCLUSIONS: Using a novel machine learning approach, we have identified an EMT-related splicing signature capable of subclassifying the most aggressive type of breast cancer, which are basal-like triple negative tumours. This proof-of-concept demonstrates that the biological knowledge acquired from cell lines can be transferred to patients data for further clinical investigation. More studies, particularly in 3D culture and organoids, will increase the accuracy of this transfer of knowledge, which will open new perspectives into the development of novel therapeutic strategies and the further identification of specific biomarkers for drug resistance and cancer relapse.

Identification of CRYAB+ KCNN3+ SOX9+ Astrocyte-Like and EGFR+ PDGFRA+ OLIG1+ Oligodendrocyte-Like Tumoral Cells in Diffuse IDH1-Mutant Gliomas and Implication of NOTCH1 Signalling in Their Genesis.

Diffuse grade II IDH-mutant gliomas are slow-growing brain tumors that progress into high-grade gliomas. They present intratumoral cell heterogeneity, and no reliable markers are available to distinguish the different cell subtypes. The molecular mechanisms underlying the formation of this cell diversity is also ill-defined. Here, we report that SOX9 and OLIG1 transcription factors, which specifically label astrocytes and oligodendrocytes in the normal brain, revealed the presence of two largely nonoverlapping tumoral populations in IDH1-mutant oligodendrogliomas and astrocytomas. Astrocyte-like SOX9+ cells additionally stained for APOE, CRYAB, ID4, KCNN3, while oligodendrocyte-like OLIG1+ cells stained for ASCL1, EGFR, IDH1, PDGFRA, PTPRZ1, SOX4, and SOX8. GPR17, an oligodendrocytic marker, was expressed by both cells. These two subpopulations appear to have distinct BMP, NOTCH1, and MAPK active pathways as stainings for BMP4, HEY1, HEY2, p-SMAD1/5 and p-ERK were higher in SOX9+ cells. We used primary cultures and a new cell line to explore the influence of NOTCH1 activation and BMP treatment on the IDH1-mutant glioma cell phenotype. This revealed that NOTCH1 globally reduced oligodendrocytic markers and IDH1 expression while upregulating APOE, CRYAB, HEY1/2, and an electrophysiologically-active Ca2+-activated apamin-sensitive K+ channel (KCNN3/SK3). This was accompanied by a reduction in proliferation. Similar effects of NOTCH1 activation were observed in nontumoral human oligodendrocytic cells, which additionally induced strong SOX9 expression. BMP treatment reduced OLIG1/2 expression and strongly upregulated CRYAB and NOGGIN, a negative regulator of BMP. The presence of astrocyte-like SOX9+ and oligodendrocyte-like OLIG1+ cells in grade II IDH1-mutant gliomas raises new questions about their role in the pathology.

Intron retention and its impact on gene expression and protein diversity: A review and a practical guide.

Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico.

iMOKA: k-mer based software to analyze large collections of sequencing data.

iMOKA (interactive multi-objective k-mer analysis) is a software that enables comprehensive analysis of sequencing data from large cohorts to generate robust classification models or explore specific genetic elements associated with disease etiology. iMOKA uses a fast and accurate feature reduction step that combines a Naïve Bayes classifier augmented by an adaptive entropy filter and a graph-based filter to rapidly reduce the search space. By using a flexible file format and distributed indexing, iMOKA can easily integrate data from multiple experiments and also reduces disk space requirements and identifies changes in transcript levels and single nucleotide variants. iMOKA is available at https://github.com/RitchieLabIGH/iMOKA and Zenodo https://doi.org/10.5281/zenodo.4008947 .

TALC: Transcript-level Aware Long-read Correction.

MOTIVATION: Long-read sequencing technologies are invaluable for determining complex RNA transcript architectures but are error-prone. Numerous 'hybrid correction' algorithms have been developed for genomic data that correct long reads by exploiting the accuracy and depth of short reads sequenced from the same sample. These algorithms are not suited for correcting more complex transcriptome sequencing data. RESULTS: We have created a novel reference-free algorithm called Transcript-level Aware Long-Read Correction (TALC) which models changes in RNA expression and isoform representation in a weighted De Bruijn graph to correct long reads from transcriptome studies. We show that transcript-level aware correction by TALC improves the accuracy of the whole spectrum of downstream RNA-seq applications and is thus necessary for transcriptome analyses that use long read technology. AVAILABILITY AND IMPLEMENTATION: TALC is implemented in C++ and available at https://github.com/lbroseus/TALC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NF90 modulates processing of a subset of human pri-miRNAs.

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

Challenges in detecting and quantifying intron retention from next generation sequencing data.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions and in disease. Here we give an overview of the different computational approaches for detecting IR events from sequencing data. We show that these are based on different biological and computational assumptions that may lead to dramatically different results. We describe the various approaches for mitigating errors in detecting intron retention and for discovering IR signatures between different conditions.

GECKO is a genetic algorithm to classify and explore high throughput sequencing data.

Comparative analysis of high throughput sequencing data between multiple conditions often involves mapping of sequencing reads to a reference and downstream bioinformatics analyses. Both of these steps may introduce heavy bias and potential data loss. This is especially true in studies where patient transcriptomes or genomes may vary from their references, such as in cancer. Here we describe a novel approach and associated software that makes use of advances in genetic algorithms and feature selection to comprehensively explore massive volumes of sequencing data to classify and discover new sequences of interest without a mapping step and without intensive use of specialized bioinformatics pipelines. We demonstrate that our approach called GECKO for GEnetic Classification using k-mer Optimization is effective at classifying and extracting meaningful sequences from multiple types of sequencing approaches including mRNA, microRNA, and DNA methylome data.

Exploring the Roles of CREBRF and TRIM2 in the Regulation of Angiogenesis by High-Density Lipoproteins.

Angiogenesis, the process of forming new blood vessels, is crucial in the physiological response to ischemia, though it can be detrimental as part of inflammation and tumorigenesis. We have previously shown that high-density lipoproteins (HDL) modulate angiogenesis in a context-specific manner via distinct classical signalling pathways, enhancing hypoxia-induced angiogenesis while suppressing inflammatory-driven angiogenesis. Whether additional novel targets exist to account for these effects are unknown. A microarray approach identified two novel genes, cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) and tripartite motif-containing protein 2 (TRIM2) that were upregulated by reconstituted HDL (rHDL). We measured CREBRF and TRIM2 expression in human coronary artery endothelial cells following incubation with rHDL and exposure to either hypoxia or an inflammatory stimulus. We found that CREBRF and TRIM2 mRNA were significantly upregulated by rHDL, particularly in response to its phospholipid component 1-palmitoyl-2-linoleoyl-phosphatidylcholine, however, protein expression was not significantly altered. Knockdown of TRIM2 impaired endothelial cell tubulogenesis in vitro in both hypoxia and inflammation, implying a necessary role in angiogenesis. Furthermore, TRIM2 knockdown attenuated rHDL-induced tubule formation in hypoxia, suggesting that it is important in mediating the pro-angiogenic action of rHDL. Our study has implications for understanding the regulation of angiogenesis in both of these pathophysiological contexts by HDL.

Identifying microRNA determinants of human myelopoiesis.

Myelopoiesis involves differentiation of hematopoietic stem cells to cellular populations that are restricted in their self-renewal capacity, beginning with the common myeloid progenitor (CMP) and leading to mature cells including monocytes and granulocytes. This complex process is regulated by various extracellular and intracellular signals including microRNAs (miRNAs). We characterised the miRNA profile of human CD34+CD38+ myeloid progenitor cells, and mature monocytes and granulocytes isolated from cord blood using TaqMan Low Density Arrays. We identified 19 miRNAs that increased in both cell types relative to the CMP and 27 that decreased. miR-125b and miR-10a were decreased by 10-fold and 100-fold respectively in the mature cells. Using in vitro granulopoietic differentiation of human CD34+ cells we show that decreases in both miR-125b and miR-10a correlate with a loss of CD34 expression and gain of CD11b and CD15 expression. Candidate target mRNAs were identified by co-incident predictions between the miRanda algorithm and genes with increased expression during differentiation. Using luciferase assays we confirmed MCL1 and FUT4 as targets of miR-125b and the transcription factor KLF4 as a target of miR-10a. Together, our data identify miRNAs with differential expression during myeloid development and reveal some relevant miRNA-target pairs that may contribute to physiological differentiation.

An NF90/NF110-mediated feedback amplification loop regulates dicer expression and controls ovarian carcinoma progression.

Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease, and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, which is embedded in DICER pre-mRNA. As miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis, whereas overexpression of miR-3173 dramatically increased metastasis in an NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.

Intron retention enhances gene regulatory complexity in vertebrates.

BACKGROUND: While intron retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution. RESULTS: Our RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of intron-retaining transcripts. We find that intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3' UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that intron-retaining genes are highly regulated post-transcriptionally. CONCLUSIONS: Our study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment.

While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression.

IRFinder: assessing the impact of intron retention on mammalian gene expression.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.

microRNA Target Prediction.

microRNAs are short RNAs that reduce gene expression by binding to their targets. Computational predictions indicate that all human genes may be regulated by microRNAs, with each microRNA possibly targeting thousands of genes. Commonly used software will produce a prohibitive number of predicted targets for each microRNA. Here I describe procedures that refine these predictions by integrating available software and expression data from experiments available online. These procedures are tailored to experiments where predicting true targets is more important than detecting all putative targets.

The Poly-cistronic miR-23-27-24 Complexes Target Endothelial Cell Junctions: Differential Functional and Molecular Effects of miR-23a and miR-23b.

The regulation of function of endothelial cell-cell junctions is fundamental in sustaining vascular integrity. The polycistronic microRNA (miR) complexes containing miR-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. We focus on miR-23a and miR-23b, and investigate the functional effects of these miRs on junctions. While miR-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with miR-23a inhibiting permeability and miR-23b inhibiting angiogenesis. Both miRs target the junctional attractive molecule (tight junction protein 2) ZO-2 and the repulsive molecule junctional adhesion molecule C (JAM-C), although the inhibition of JAM-C by miR-23a is more profound than by miR-23b. The difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of miR-23b in the 3'UTR of JAM-C restores identity. We also show that the pattern of expression of miR-23a and miR-23b and their targets are different. Thus, the paralogues miR-23a and miR-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the miRs. This work exposes a hitherto unappreciated complexity in therapeutically targeting miRs.

HIF-1α and rapamycin act as gerosuppressant in multiple myeloma cells upon genotoxic stress.

Multiple myeloma (MM) is still an incurable hematological malignancy. Despite recent progress due to new anti-myeloma agents, the pathology is characterized by a high frequency of de novo or acquired resistance. Delineating the mechanisms of MM resistance is essential for therapeutic advances. We previously showed that long-term genotoxic stress induces the establishment of a senescence-associated secretory phenotype, a pro-inflammatory response that favors the emergence of cells with cancer stem-like properties. Here, we studied the short-term response of MM cells following treatment with various DNA damaging agents such as the energetic C-ion irradiation. MM cells are highly resistant to all treatments and do not enter apoptosis after they arrest cycling at the G2 phase. Although the DNA damage response pathway was activated, DNA breaks remained chronically in damaged MM cells. We found, using a transcriptomic approach that RAD50, a major DNA repair gene was downregulated early after genotoxic stress. In two gerosuppression situations: induction of hypoxia and inhibition of the mammalian target of rapamycin (mTOR) pathway, we observed, after the treatment with a DNA damaging agent, a normalization of RAD50 expression concomitant with the absence of cell cycle arrest. We propose that combining inhibitors of mTOR with genotoxic agents could avoid MM cells to senesce and secrete pro-inflammatory factors responsible for cancer stem-like cell emergence and, in turn, relapse of MM patients.

Beliefs about the causes and cures of depression.

BACKGROUND: This study used attitude statement and vignette methodology to examine a mixed British sample's belies about the causes and consequences of depression. AIMS: To test whether the group would recognise both vignettes with having depression and that the favoured cure would be Psychotherapy/Talking Cure. METHOD: In all, 320 adults completed a two-part questionnaire. In the first part, they were given two vignettes describing a 30-year-old female and a 45-year-old male both with depression. They were asked what they thought (if anything) was wrong with the person and how they could best be helped. In the second part, they completed two questionnaires, one which lists 47 possible causes and the other 48 possible treatments for depression. RESULTS: Most participants 'diagnosed' depression for the two vignettes although they chose very different terms and offered a variety of 'cures', including medication and counselling. The questionnaires about cause and cure factored into seven interpretable factors which were logically correlated. A series of regressions showed that sex, age, media interest, political beliefs, experience with depression and other mental illnesses as well as having known of others diagnosed with depression predicted different beliefs about the causes and cures of depression. CONCLUSION: People have a detailed and multidimensional view of the causes and cures for depression which is systematically related to each other.