RNA-Based Strategies for Cancer Therapy: In Silico Design and Evaluation of ASOs for Targeted Exon Skipping.

Precision medicine in oncology has made significant progress in recent years by approving drugs that target specific genetic mutations. However, many cancer driver genes remain challenging to pharmacologically target ("undruggable"). To tackle this issue, RNA-based methods like antisense oligonucleotides (ASOs) that induce targeted exon skipping (ES) could provide a promising alternative. In this work, a comprehensive computational procedure is presented, focused on the development of ES-based cancer treatments. The procedure aims to produce specific protein variants, including inactive oncogenes and partially restored tumor suppressors. This novel computational procedure encompasses target-exon selection, in silico prediction of ES products, and identification of the best candidate ASOs for further experimental validation. The method was effectively employed on extensively mutated cancer genes, prioritized according to their suitability for ES-based interventions. Notable genes, such as NRAS and VHL, exhibited potential for this therapeutic approach, as specific target exons were identified and optimal ASO sequences were devised to induce their skipping. To the best of our knowledge, this is the first computational procedure that encompasses all necessary steps for designing ASO sequences tailored for targeted ES, contributing with a versatile and innovative approach to addressing the challenges posed by undruggable cancer driver genes and beyond.

The Hsp70 chaperone is a major player in stress-induced transposable element activation.

Previous studies have shown that heat shock stress may activate transposable elements (TEs) in Drosophila and other organisms. Such an effect depends on the disruption of a chaperone complex that is normally involved in biogenesis of Piwi-interacting RNAs (piRNAs), the largest class of germline-enriched small noncoding RNAs implicated in the epigenetic silencing of TEs. However, a satisfying picture of how chaperones could be involved in repressing TEs in germ cells is still unknown. Here we show that, in Drosophila, heat shock stress increases the expression of TEs at a posttranscriptional level by affecting piRNA biogenesis through the action of the inducible chaperone Hsp70. We found that stress-induced TE activation is triggered by an interaction of Hsp70 with the Hsc70-Hsp90 complex and other factors all involved in piRNA biogenesis in both ovaries and testes. Such interaction induces a displacement of all such factors to the lysosomes, resulting in a functional collapse of piRNA biogenesis. This mechanism has clear evolutionary implications. In the presence of drastic environmental changes, Hsp70 plays a key dual role in increasing both the survival probability of individuals and the genetic variability in their germ cells. The consequent increase of genetic variation in a population potentiates evolutionary plasticity and evolvability.

A Plasma Circular RNA Profile Differentiates Subjects with Alzheimer's Disease and Mild Cognitive Impairment from Healthy Controls.

The most frequently used biomarkers to support the diagnosis of Alzheimer's Disease (AD) are Aß42, total-Tau, and phospho-tau protein levels in CSF. Moreover, magnetic resonance imaging is used to assess hippocampal atrophy, 18F-FDG PET to identify abnormal brain metabolism, and PET imaging for amyloid deposition. These tests are rather complex and invasive and not easily applicable to clinical practice. Circulating non-coding RNAs, which are inherently stable and easy to manage, have been reported as promising biomarkers for central nervous system conditions. Recently, circular RNAs (circRNAs) as a novel class of ncRNAs have gained attention. We carried out a pilot study on five participants with AD and five healthy controls (HC) investigating circRNAs by Arraystar Human Circular RNA Microarray V2.0. Among them, 26 circRNAs were differentially expressed (FC ≥ 1.5, p < 0.05) in participants with AD compared to HC. From a top 10 of differentially expressed circRNAs, a validation study was carried out on four up-regulated (hsa_circRNA_050263, hsa_circRNA_403959, hsa_circRNA_003022, hsa_circRNA_100837) and two down-regulated (hsa_circRNA_102049, hsa_circRNA_102619) circRNAs in a larger population. Moreover, five subjects with mild cognitive impairment (MCI) were investigated. The analysis confirmed the upregulation of hsa_circRNA_050263, hsa_circRNA_403959, and hsa_circRNA_003022 both in subjects with AD and in MCI compared to HCs. We also investigated all microRNAs potentially interacting with the studied circRNAs. The GO enrichment analysis shows they are involved in the development of the nervous system, and in the cellular response to nerve growth factor stimuli, protein phosphorylation, apoptotic processes, and inflammation pathways, all of which are processes related to the pathology of AD.

Ten simple rules for making training materials FAIR.

Everything we do today is becoming more and more reliant on the use of computers. The field of biology is no exception; but most biologists receive little or no formal preparation for the increasingly computational aspects of their discipline. In consequence, informal training courses are often needed to plug the gaps; and the demand for such training is growing worldwide. To meet this demand, some training programs are being expanded, and new ones are being developed. Key to both scenarios is the creation of new course materials. Rather than starting from scratch, however, it's sometimes possible to repurpose materials that already exist. Yet finding suitable materials online can be difficult: They're often widely scattered across the internet or hidden in their home institutions, with no systematic way to find them. This is a common problem for all digital objects. The scientific community has attempted to address this issue by developing a set of rules (which have been called the Findable, Accessible, Interoperable and Reusable [FAIR] principles) to make such objects more findable and reusable. Here, we show how to apply these rules to help make training materials easier to find, (re)use, and adapt, for the benefit of all.

A framework to assess the quality and impact of bioinformatics training across ELIXIR.

ELIXIR is a pan-European intergovernmental organisation for life science that aims to coordinate bioinformatics resources in a single infrastructure across Europe; bioinformatics training is central to its strategy, which aims to develop a training community that spans all ELIXIR member states. In an evidence-based approach for strengthening bioinformatics training programmes across Europe, the ELIXIR Training Platform, led by the ELIXIR EXCELERATE Quality and Impact Assessment Subtask in collaboration with the ELIXIR Training Coordinators Group, has implemented an assessment strategy to measure quality and impact of its entire training portfolio. Here, we present ELIXIR's framework for assessing training quality and impact, which includes the following: specifying assessment aims, determining what data to collect in order to address these aims, and our strategy for centralised data collection to allow for ELIXIR-wide analyses. In addition, we present an overview of the ELIXIR training data collected over the past 4 years. We highlight the importance of a coordinated and consistent data collection approach and the relevance of defining specific metrics and answer scales for consortium-wide analyses as well as for comparison of data across iterations of the same course.

A Census and Categorization Method of Epitranscriptomic Marks.

In the past few years, thorough investigation of chemical modifications operated in the cells on ribonucleic acid (RNA) molecules is gaining momentum. This new field of research has been dubbed "epitranscriptomics", in analogy to best-known epigenomics, to stress the potential of ensembles of RNA modifications to constitute a post-transcriptional regulatory layer of gene expression orchestrated by writer, reader, and eraser RNA-binding proteins (RBPs). In fact, epitranscriptomics aims at identifying and characterizing all functionally relevant changes involving both non-substitutional chemical modifications and editing events made to the transcriptome. Indeed, several types of RNA modifications that impact gene expression have been reported so far in different species of cellular RNAs, including ribosomal RNAs, transfer RNAs, small nuclear RNAs, messenger RNAs, and long non-coding RNAs. Supporting functional relevance of this largely unknown regulatory mechanism, several human diseases have been associated directly to RNA modifications or to RBPs that may play as effectors of epitranscriptomic marks. However, an exhaustive epitranscriptome's characterization, aimed to systematically classify all RNA modifications and clarify rules, actors, and outcomes of this promising regulatory code, is currently not available, mainly hampered by lack of suitable detecting technologies. This is an unfortunate limitation that, thanks to an unprecedented pace of technological advancements especially in the sequencing technology field, is likely to be overcome soon. Here, we review the current knowledge on epitranscriptomic marks and propose a categorization method based on the reference ribonucleotide and its rounds of modifications ("stages") until reaching the given modified form. We believe that this classification scheme can be useful to coherently organize the expanding number of discovered RNA modifications.

Genome-wide identification of direct HBx genomic targets.

BACKGROUND: The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. RESULTS: ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. CONCLUSIONS: Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

3USS: a web server for detecting alternative 3'UTRs from RNA-seq experiments.

UNLABELLED: Protein-coding genes with multiple alternative polyadenylation sites can generate mRNA 3'UTR sequences of different lengths, thereby causing the loss or gain of regulatory elements, which can affect stability, localization and translation efficiency. 3USS is a web-server developed with the aim of giving experimentalists the possibility to automatically identify alternative 3 ': UTRs (shorter or longer with respect to a reference transcriptome), an option that is not available in standard RNA-seq data analysis procedures. The tool reports as putative novel the 3 ': UTRs not annotated in available databases. Furthermore, if data from two related samples are uploaded, common and specific alternative 3 ': UTRs are identified and reported by the server. AVAILABILITY AND IMPLEMENTATION: 3USS is freely available at http://www.biocomputing.it/3uss_server.

Interleukin-8 in Colorectal Cancer: A Systematic Review and Meta-Analysis of Its Potential Role as a Prognostic Biomarker.

Among soluble actors that have emerged as druggable factors, the chemokine interleukin-8 (IL-8) has emerged as a possible determinant of response to immunotherapy and targeted treatment in several cancer types; however, its prognostic/predictive role in colorectal cancer (CRC) remains to be established. We: (i) conducted a systematic review of published literature on IL-8 expression in CRC; (ii) searched public transcriptomics databases; (iii) investigated IL-8 expression, by tumor and infiltrating cells, in a series of CRC samples; and (iv) carried out a meta-analysis of published literature correlating IL-8 expression and CRC prognosis. IL-8 possesses an important role as a mediator of the bidirectional crosstalk between tumor/stromal cells. Transcriptomic analysis indicated that specific IL-8 transcripts were significantly overexpressed in CRC compared to normal colon mucosa. Moreover, in our series we observed a statistically significant correlation between PTEN-loss and IL-8 expression by infiltrating mononuclear and tumor cells. In total, 12 papers met our meta-analysis inclusion criteria, demonstrating that high IL-8 levels significantly correlated with shorter overall survival and progression-free survival. Sensitivity analysis demonstrated a highly significant correlation with outcome for circulating, but not for tissue-detected, IL-8. IL-8 is overexpressed in CRC tissues and differentially produced by tumor or stromal components depending on CRC genetic background. Moreover, circulating IL-8 represents a strong prognostic factor in CRC, suggesting its use in the refining of prognostic CRC assessment and potentially the tailoring of therapeutic strategies in individual CRC patients.

PICMI: mapping point mutations on genomes.

MOTIVATION: Several international collaborations and local projects are producing extensive catalogues of genomic variations that are supplementing existing collections such as the OMIM catalogue. The flood of this type of data will keep increasing and, especially, it will be relevant to a wider user base, including not only molecular biologists, geneticists and bioinformaticians, but also clinical researchers. Mapping the observed variations, sometimes only described at the amino acid level, on a genome, identifying whether they affect a gene and-if so-whether they also affect different isoforms of the same gene, is a time consuming and often frustrating task. RESULTS: The PICMI server is an easy to use tool for quickly mapping one or more amino acid or nucleotide variations on a genome and its products, including alternatively spliced isoforms. AVAILABILITY: The server is available at www.biocomputing.it/picmi.

AI applications in functional genomics.

We review the current applications of artificial intelligence (AI) in functional genomics. The recent explosion of AI follows the remarkable achievements made possible by "deep learning", along with a burst of "big data" that can meet its hunger. Biology is about to overthrow astronomy as the paradigmatic representative of big data producer. This has been made possible by huge advancements in the field of high throughput technologies, applied to determine how the individual components of a biological system work together to accomplish different processes. The disciplines contributing to this bulk of data are collectively known as functional genomics. They consist in studies of: i) the information contained in the DNA (genomics); ii) the modifications that DNA can reversibly undergo (epigenomics); iii) the RNA transcripts originated by a genome (transcriptomics); iv) the ensemble of chemical modifications decorating different types of RNA transcripts (epitranscriptomics); v) the products of protein-coding transcripts (proteomics); and vi) the small molecules produced from cell metabolism (metabolomics) present in an organism or system at a given time, in physiological or pathological conditions. After reviewing main applications of AI in functional genomics, we discuss important accompanying issues, including ethical, legal and economic issues and the importance of explainability.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Funding: Telethon Italia (grant #GGP12116).

Importin-ß/karyopherin-ß1 modulates mitotic microtubule function and taxane sensitivity in cancer cells via its nucleoporin-binding region.

The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.

Foxm1 controls a pro-stemness microRNA network in neural stem cells.

Cerebellar neural stem cells (NSCs) require Hedgehog-Gli (Hh-Gli) signalling for their maintenance and Nanog expression for their self-renewal. To identify novel molecular features of this regulatory pathway, we used next-generation sequencing technology to profile mRNA and microRNA expression in cerebellar NSCs, before and after induced differentiation (Diff-NSCs). Genes with higher transcript levels in NSCs (vs. Diff-NSCs) included Foxm1, which proved to be directly regulated by Gli and Nanog. Foxm1 in turn regulated several microRNAs that were overexpressed in NSCs: miR-130b, miR-301a, and members of the miR-15~16 and miR-17~92 clusters and whose knockdown significantly impaired the neurosphere formation ability. Our results reveal a novel Hh-Gli-Nanog-driven Foxm1-microRNA network that controls the self-renewal capacity of NSCs.

The loss of ATP2C1 impairs the DNA damage response and induces altered skin homeostasis: Consequences for epidermal biology in Hailey-Hailey disease.

Mutation of the Golgi Ca(2+)-ATPase ATP2C1 is associated with deregulated calcium homeostasis and altered skin function. ATP2C1 mutations have been identified as having a causative role in Hailey-Hailey disease, an autosomal-dominant skin disorder. Here, we identified ATP2C1 as a crucial regulator of epidermal homeostasis through the regulation of oxidative stress. Upon ATP2C1 inactivation, oxidative stress and Notch1 activation were increased in cultured human keratinocytes. Using RNA-seq experiments, we found that the DNA damage response (DDR) was consistently down-regulated in keratinocytes derived from the lesions of patients with Hailey-Hailey disease. Although oxidative stress activates the DDR, ATP2C1 inactivation down-regulates DDR gene expression. We showed that the DDR response was a major target of oxidative stress-induced Notch1 activation. Here, we show that this activation is functionally important because early Notch1 activation in keratinocytes induces keratinocyte differentiation and represses the DDR. These results indicate that an ATP2C1/NOTCH1 axis might be critical for keratinocyte function and cutaneous homeostasis, suggesting a plausible model for the pathological features of Hailey-Hailey disease.

Coding potential of the products of alternative splicing in human.

BACKGROUND: Analysis of the human genome has revealed that as much as an order of magnitude more of the genomic sequence is transcribed than accounted for by the predicted and characterized genes. A number of these transcripts are alternatively spliced forms of known protein coding genes; however, it is becoming clear that many of them do not necessarily correspond to a functional protein. RESULTS: In this study we analyze alternative splicing isoforms of human gene products that are unambiguously identified by mass spectrometry and compare their properties with those of isoforms of the same genes for which no peptide was found in publicly available mass spectrometry datasets. We analyze them in detail for the presence of uninterrupted functional domains, active sites as well as the plausibility of their predicted structure. We report how well each of these strategies and their combination can correctly identify translated isoforms and derive a lower limit for their specificity, that is, their ability to correctly identify non-translated products. CONCLUSIONS: The most effective strategy for correctly identifying translated products relies on the conservation of active sites, but it can only be applied to a small fraction of isoforms, while a reasonably high coverage, sensitivity and specificity can be achieved by analyzing the presence of non-truncated functional domains. Combining the latter with an assessment of the plausibility of the modeled structure of the isoform increases both coverage and specificity with a moderate cost in terms of sensitivity.