Intron retention: importance, challenges, and opportunities.

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

Recent advances in cancer fusion transcript detection.

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts.

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

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

Prospects and challenges of cancer systems medicine: from genes to disease networks.

It is becoming evident that holistic perspectives toward cancer are crucial in deciphering the overwhelming complexity of tumors. Single-layer analysis of genome-wide data has greatly contributed to our understanding of cellular systems and their perturbations. However, fundamental gaps in our knowledge persist and hamper the design of effective interventions. It is becoming more apparent than ever, that cancer should not only be viewed as a disease of the genome but as a disease of the cellular system. Integrative multilayer approaches are emerging as vigorous assets in our endeavors to achieve systemic views on cancer biology. Herein, we provide a comprehensive review of the approaches, methods and technologies that can serve to achieve systemic perspectives of cancer. We start with genome-wide single-layer approaches of omics analyses of cellular systems and move on to multilayer integrative approaches in which in-depth descriptions of proteogenomics and network-based data analysis are provided. Proteogenomics is a remarkable example of how the integration of multiple levels of information can reduce our blind spots and increase the accuracy and reliability of our interpretations and network-based data analysis is a major approach for data interpretation and a robust scaffold for data integration and modeling. Overall, this review aims to increase cross-field awareness of the approaches and challenges regarding the omics-based study of cancer and to facilitate the necessary shift toward holistic approaches.

The application of long-read sequencing in clinical settings.

Long-read DNA sequencing technologies have been rapidly evolving in recent years, and their ability to assess large and complex regions of the genome makes them ideal for clinical applications in molecular diagnosis and therapy selection, thereby providing a valuable tool for precision medicine. In the third-generation sequencing duopoly, Oxford Nanopore Technologies and Pacific Biosciences work towards increasing the accuracy, throughput, and portability of long-read sequencing methods while trying to keep costs low. These trades have made long-read sequencing an attractive tool for use in research and clinical settings. This article provides an overview of current clinical applications and limitations of long-read sequencing and explores its potential for point-of-care testing and health care in remote settings.

Increased chromatin accessibility facilitates intron retention in specific cell differentiation states.

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.

A signal-seeking Phase 2 study of olaparib and durvalumab in advanced solid cancers with homologous recombination repair gene alterations.

PURPOSE: To determine the safety and efficacy of PARP plus PD-L1 inhibition (olaparib + durvalumab, O + D) in patients with advanced solid, predominantly rare cancers harbouring homologous recombination repair (HRR) defects. PATIENTS AND METHODS: In total, 48 patients were treated with O + D, 16 with BRCA1/2 alterations (group 1) and 32 with other select HRR alterations (group 2). Overall, 32 (66%) patients had rare or less common cancers. The primary objective of this single-arm Phase II trial was a progression-free survival rate at 6 months (PFS6). Post hoc exploratory analyses were conducted on archival tumour tissue and serial bloods. RESULTS: The PFS6 rate was 35% and 38% with durable objective tumour responses (OTR) in 3(19%) and 3(9%) in groups 1 and 2, respectively. Rare cancers achieving an OTR included cholangiocarcinoma, perivascular epithelioid cell (PEComa), neuroendocrine, gallbladder and endometrial cancer. O + D was safe, with five serious adverse events related to the study drug(s) in 3 (6%) patients. A higher proportion of CD38 high B cells in the blood and higher CD40 expression in tumour was prognostic of survival. CONCLUSIONS: O + D demonstrated no new toxicity concerns and yielded a clinically meaningful PFS6 rate and durable OTRs across several cancers with HRR defects, including rare cancers.

CTCF as a regulator of alternative splicing: new tricks for an old player.

Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the 'master weaver of the genome'. Given its multiple connections, further characterization of CTCF's emerging role in splicing regulation might extend its functional repertoire towards a 'conductor of the splicing orchestra'.

Towards resolution of the intron retention paradox in breast cancer.

BACKGROUND: After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue. METHODS: In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR. RESULTS: Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa. CONCLUSIONS: Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

Structure-function relationships explain CTCF zinc finger mutation phenotypes in cancer.

CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure-function relationships.

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

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

The Role of MicroRNAs in Cancer Biology and Therapy from a Systems Biology Perspective.

Since the discovery of microRNAs (miRNAs) in Caenorhabditis elegans, our understanding of their cellular function has progressed continuously. Today, we have a good understanding of miRNA-mediated gene regulation, miRNA-mediated cross talk between genes including competing endogenous RNAs, and miRNA-mediated signaling transduction both in normal human physiology and in diseases.Besides, these noncoding RNAs have shown their value for clinical applications, especially in an oncological context. They can be used as reliable biomarkers for cancer diagnosis and prognosis and attract increasing attention as potential therapeutic targets. Many achievements made in the miRNA field are based on joint efforts from computational and molecular biologists. Systems biology approaches, which integrate computational and experimental methods, have played a fundamental role in uncovering the cellular functions of miRNAs.In this chapter, we review and discuss the role of miRNAs in oncology from a system biology perspective. We first describe biological facts about miRNA genetics and function. Next, we discuss the role of miRNAs in cancer progression and review the application of miRNAs in cancer diagnostics and therapy. Finally, we elaborate on the role that miRNAs play in cancer gene regulatory networks. Taken together, we emphasize the importance of systems biology approaches in our continued efforts to study miRNA cancer regulation.

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

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

Systems biology-based investigation of cooperating microRNAs as monotherapy or adjuvant therapy in cancer.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression by suppressing mRNA translation and reducing mRNA stability. A miRNA can potentially bind many mRNAs, thereby affecting the expression of oncogenes and tumor suppressor genes as well as the activity of whole pathways. The promise of miRNA therapeutics in cancer is to harness this evolutionarily conserved mechanism for the coordinated regulation of gene expression, and thus restoring a normal cell phenotype. However, the promiscuous binding of miRNAs can provoke unwanted off-target effects, which are usually caused by high-dose single-miRNA treatments. Thus, it is desirable to develop miRNA therapeutics with increased specificity and efficacy. To achieve that, we propose the concept of miRNA cooperativity in order to exert synergistic repression on target genes, thus lowering the required total amount of miRNAs. We first review miRNA therapies in clinical application. Next, we summarize the knowledge on the molecular mechanism and biological function of miRNA cooperativity and discuss its application in cancer therapies. We then propose and discuss a systems biology approach to investigate miRNA cooperativity for the clinical setting. Altogether, we point out the potential of miRNA cooperativity to reduce off-target effects and to complement conventional, targeted, or immune-based therapies for cancer.

The changing paradigm of intron retention: regulation, ramifications and recipes.

Intron retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, insects and viruses. It was generally assumed that mis-splicing, leading to the retention of introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant.

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells.

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein-protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

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

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

Exosomal lncRNAs and cancer: connecting the missing links.

Motivation: Extracellular vesicles (EVs), including exosomes and microvesicles, are potent and clinically valuable tools for early diagnosis, prognosis and potentially the targeted treatment of cancer. The content of EVs is closely related to the type and status of the EV-secreting cell. Circulating exosomes are a source of stable RNAs including mRNAs, microRNAs and long non-coding RNAs (lncRNAs). Results: This review outlines the links between EVs, lncRNAs and cancer. We highlight communication networks involving the tumor microenvironment, the immune system and metastasis. We show examples supporting the value of exosomal lncRNAs as cancer biomarkers and therapeutic targets. We demonstrate how a system biology approach can be used to model cell-cell communication via exosomal lncRNAs and to simulate effects of therapeutic interventions. In addition, we introduce algorithms and bioinformatics resources for the discovery of tumor-specific lncRNAs and tools that are applied to determine exosome content and lncRNA function. Finally, this review provides a comprehensive collection and guide to databases for exosomal lncRNAs. Supplementary information: Supplementary data are available at Bioinformatics online.

The RNA world in the 21st century-a systems approach to finding non-coding keys to clinical questions.

There was evidence that RNAs are a functionally rich class of molecules not only since the arrival of the next-generation sequencing technology. Non-coding RNAs (ncRNA) could be the key to accelerated diagnosis and enhanced prediction of disease and therapy outcomes as well as the design of advanced therapeutic strategies to overcome yet unsatisfactory approaches.In this review, we discuss the state of the art in RNA systems biology with focus on the application in the systems biomedicine field. We propose guidelines for analysing the role of microRNAs and long non-coding RNAs in human pathologies. We introduce RNA expression profiling and network approaches for the identification of stable and effective RNomics-based biomarkers, providing insights into the role of ncRNAs in disease regulation. Towards this, we discuss ways to model the dynamics of gene regulatory networks and signalling pathways that involve ncRNAs. We also describe data resources and computational methods for finding putative mechanisms of action of ncRNAs. Finally, we discuss avenues for the computer-aided design of novel RNA-based therapeutics.

Personalized cancer immunotherapy using Systems Medicine approaches.

The immune system is by definition multi-scale because it involves biochemical networks that regulate cell fates across cell boundaries, but also because immune cells communicate with each other by direct contact or through the secretion of local or systemic signals. Furthermore, tumor and immune cells communicate, and this interaction is affected by the tumor microenvironment. Altogether, the tumor-immunity interaction is a complex multi-scale biological system whose analysis requires a systemic view to succeed in developing efficient immunotherapies for cancer and immune-related diseases. In this review we discuss the necessity and the structure of a systems medicine approach for the design of anticancer immunotherapies. We support the idea that the approach must be a combination of algorithms and methods from bioinformatics and patient-data-driven mathematical models conceived to investigate the role of clinical interventions in the tumor-immunity interaction. For each step of the integrative approach proposed, we review the advancement with respect to the computational tools and methods available, but also successful case studies. We particularized our idea for the case of identifying novel tumor-associated antigens and therapeutic targets by integration of patient's immune and tumor profiling in case of aggressive melanoma.