Functional identification of cis-regulatory long noncoding RNAs at controlled false discovery rates.

A key attribute of some long noncoding RNAs (lncRNAs) is their ability to regulate expression of neighbouring genes in cis. However, such 'cis-lncRNAs' are presently defined using ad hoc criteria that, we show, are prone to false-positive predictions. The resulting lack of cis-lncRNA catalogues hinders our understanding of their extent, characteristics and mechanisms. Here, we introduce TransCistor, a framework for defining and identifying cis-lncRNAs based on enrichment of targets amongst proximal genes. TransCistor's simple and conservative statistical models are compatible with functionally defined target gene maps generated by existing and future technologies. Using transcriptome-wide perturbation experiments for 268 human and 134 mouse lncRNAs, we provide the first large-scale survey of cis-lncRNAs. Known cis-lncRNAs are correctly identified, including XIST, LINC00240 and UMLILO, and predictions are consistent across analysis methods, perturbation types and independent experiments. We detect cis-activity in a minority of lncRNAs, primarily involving activators over repressors. Cis-lncRNAs are detected by both RNA interference and antisense oligonucleotide perturbations. Mechanistically, cis-lncRNA transcripts are observed to physically associate with their target genes and are weakly enriched with enhancer elements. In summary, TransCistor establishes a quantitative foundation for cis-lncRNAs, opening a path to elucidating their molecular mechanisms and biological significance.

BRD2 and BRD3 genes independently evolved RNA structures to control unproductive splicing.

The mammalian BRD2 and BRD3 genes encode structurally related proteins from the bromodomain and extraterminal domain protein family. The expression of BRD2 is regulated by unproductive splicing upon inclusion of exon 3b, which is located in the region encoding a bromodomain. Bioinformatic analysis indicated that BRD2 exon 3b inclusion is controlled by a pair of conserved complementary regions (PCCR) located in the flanking introns. Furthermore, we identified a highly conserved element encoding a cryptic poison exon 5b and a previously unknown PCCR in the intron between exons 5 and 6 of BRD3, however, outside of the homologous bromodomain. Minigene mutagenesis and blockage of RNA structure by antisense oligonucleotides demonstrated that RNA structure controls the rate of inclusion of poison exons. The patterns of BRD2 and BRD3 expression and splicing show downregulation upon inclusion of poison exons, which become skipped in response to transcription elongation slowdown, further confirming a role of PCCRs in unproductive splicing regulation. We conclude that BRD2 and BRD3 independently acquired poison exons and RNA structures to dynamically control unproductive splicing. This study describes a convergent evolution of regulatory unproductive splicing mechanisms in these genes, providing implications for selective modulation of their expression in therapeutic applications.

Long-range RNA structures in the human transcriptome beyond evolutionarily conserved regions.

RNA structure has been increasingly recognized as a critical player in the biogenesis and turnover of many transcripts classes. In eukaryotes, the prediction of RNA structure by thermodynamic modeling meets fundamental limitations due to the large sizes and complex, discontinuous organization of eukaryotic genes. Signatures of functional RNA structures can be found by detecting compensatory substitutions in homologous sequences, but a comparative approach is applicable only within conserved sequence blocks. Here, we developed a computational pipeline called PHRIC, which is not limited to conserved regions and relies on RNA contacts derived from RNA in situ conformation sequencing (RIC-seq) experiments. It extracts pairs of short RNA fragments surrounded by nested clusters of RNA contacts and predicts long, nearly perfect complementary base pairings formed between these fragments. In application to a panel of RIC-seq experiments in seven human cell lines, PHRIC predicted ~12,000 stable long-range RNA structures with equilibrium free energy below -15 kcal/mol, the vast majority of which fall outside of regions annotated as conserved among vertebrates. These structures, nevertheless, show some level of sequence conservation and remarkable compensatory substitution patterns in other clades. Furthermore, we found that introns have a higher propensity to form stable long-range RNA structures between each other, and moreover that RNA structures tend to concentrate within the same intron rather than connect adjacent introns. These results for the first time extend the application of proximity ligation assays to RNA structure prediction beyond conserved regions.

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation.

Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3'-ends have uncovered widespread intronic polyadenylation (IPA) in normal and disease conditions, where it is currently attributed to stochastic variations in the pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific expression of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines revealed that APA events are more abundant in introns than in exons. While the rate of IPA in composite terminal exons and skipped terminal exons expectedly correlates with splicing, we observed a considerable fraction of IPA events that lack AS support and attributed them to spliced polyadenylated introns (SPI). We hypothesize that SPIs represent transient byproducts of a dynamic coupling between APA and AS, in which the spliceosome removes the intron while it is being cleaved and polyadenylated. These findings indicate that cotranscriptional pre-mRNA splicing could serve as a rescue mechanism to suppress premature transcription termination at intronic polyadenylation sites.

RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

Tissue-specific regulation of gene expression via unproductive splicing.

Eukaryotic gene expression is regulated post-transcriptionally by a mechanism called unproductive splicing, in which mRNA is triggered to degrade by the nonsense-mediated decay (NMD) pathway as a result of regulated alternative splicing (AS). Only a few dozen unproductive splicing events (USEs) are currently documented, and many more remain to be identified. Here, we analyzed RNA-seq experiments from the Genotype-Tissue Expression (GTEx) Consortium to identify USEs, in which an increase in the NMD isoform splicing rate is accompanied by tissue-specific down-regulation of the host gene. To characterize RNA-binding proteins (RBPs) that regulate USEs, we superimposed these results with RBP footprinting data and experiments on the response of the transcriptome to the perturbation of expression of a large panel of RBPs. Concordant tissue-specific changes between the expression of RBP and USE splicing rate revealed a high-confidence regulatory network including 27 tissue-specific USEs with strong evidence of RBP binding. Among them, we found previously unknown PTBP1-controlled events in the DCLK2 and IQGAP1 genes, for which we confirmed the regulatory effect using small interfering RNA (siRNA) knockdown experiments in the A549 cell line. In sum, we present a transcriptomic pipeline that allows the identification of tissue-specific USEs, potentially many more than were reported here using stringent filters.

Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs.

Significant alterations in signaling pathways and transcriptional regulatory programs together represent major hallmarks of many cancers. These, among all, include the reactivation of stemness, which is registered by the expression of pathways that are active in the embryonic stem cells (ESCs). Here, we assembled gene sets that reflect the stemness and proliferation signatures and used them to analyze a large panel of RNA-seq data from The Cancer Genome Atlas (TCGA) Consortium in order to specifically assess the expression of stemness-related and proliferation-related genes across a collection of different tumor types. We introduced a metric that captures the collective similarity of the expression profile of a tumor to that of ESCs, which showed that stemness and proliferation signatures vary greatly between different tumor types. We also observed a high degree of intertumoral heterogeneity in the expression of stemness- and proliferation-related genes, which was associated with increased hazard ratios in a fraction of tumors and mirrored by high intratumoral heterogeneity and a remarkable stemness capacity in metastatic lesions across cancer cells in single cell RNA-seq datasets. Taken together, these results indicate that the expression of stemness signatures is highly heterogeneous and cannot be used as a universal determinant of cancer. This calls into question the universal validity of diagnostic tests that are based on stem cell markers.

An extended catalogue of tandem alternative splice sites in human tissue transcriptomes.

Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 46k miSS with sufficient read support, 9k (20%) are significantly expressed above the expected noise level, and among them 2.5k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP), tissue-specific expression of miSS, and miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. The analysis of human primary cell transcriptomes suggested that both tissue-specific and cell-type-specific factors contribute to the regulation of miSS expression. More than 20% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 15% of maSS and not more than 54% of tissue-specific miSS are noisy, while the proportion of noisy splice sites among non-significantly expressed miSS is above 63%.

Conserved long-range base pairings are associated with pre-mRNA processing of human genes.

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

Multiple competing RNA structures dynamically control alternative splicing in the human ATE1 gene.

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.

A limited set of transcriptional programs define major cell types.

We have produced RNA sequencing data for 53 primary cells from different locations in the human body. The clustering of these primary cells reveals that most cells in the human body share a few broad transcriptional programs, which define five major cell types: epithelial, endothelial, mesenchymal, neural, and blood cells. These act as basic components of many tissues and organs. Based on gene expression, these cell types redefine the basic histological types by which tissues have been traditionally classified. We identified genes whose expression is specific to these cell types, and from these genes, we estimated the contribution of the major cell types to the composition of human tissues. We found this cellular composition to be a characteristic signature of tissues and to reflect tissue morphological heterogeneity and histology. We identified changes in cellular composition in different tissues associated with age and sex, and found that departures from the normal cellular composition correlate with histological phenotypes associated with disease.

Urinary biomarkers of latent inflammation and fibrosis in children with vesicoureteral reflux.

PURPOSE: To investigate the urinary levels of TGF-ß1, VEGF, and MCP-1 as potential biomarkers of latent inflammation and fibrosis in the kidney before and 6 months after correction of vesicoureteral reflux (VUR) in children. METHODS: A total of 88 patients (mean age 26 months) with VUR were divided into three groups: group A-patients with grades II-III VUR, conservative treatment; group B-patients with grades III-V VUR, endoscopic correction of VUR; group C-patients with grades III-V VUR, ureteral reimplantation after failed endoscopic correction. Control group included 20 healthy children. Biomarker levels were measured by ELISA. 99mTc-DMSA scintigraphy and renal histology were performed if possible. RESULTS: At admission, TGF-ß1 was close to control in all study groups, VEGF increased with severity of the disease, and MCP-1 increased in group C. Six months after correction of VUR, despite clinical and laboratory improvement, TGF-ß1 and MCP-1 increased while VEGF decreased compared to the admission values in all groups; no amelioration of renal scarring was detected either by 99mTc-DMSA scintigraphy or renal histology. CONCLUSION: The results support our hypothesis that successful correction of VUR is not sufficient to stop or reduce the latent inflammatory and fibrotic processes that have already started in the kidney regardless of the reflux grade and treatment option. Measuring the urinary levels of TGF-ß1, VEGF, and MCP-1 may aid in the development of non-invasive, pathophysiologically relevant approach to diagnosis and monitoring of kidney injury and fibrosis in children with VUR.

Re-annotation of 191 developmental and epileptic encephalopathy-associated genes unmasks de novo variants in SCN1A.

The developmental and epileptic encephalopathies (DEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for a lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 putative transcript models. Our annotations increase the transcriptional 'footprint' of these genes by over 674 kb. Using SCN1A as a case study, due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome or a similar phenotype with a panel of exon sequences representing eight established genes and identified two de novo SCN1A variants that now - through improved gene annotation - are ascribed to residing among our exons. These two (from 122 screened people, 1.6%) molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously classified SCN1A intronic Dravet syndrome-associated variant that now lies within a deeply conserved exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders.

Circular exonic RNAs: When RNA structure meets topology.

Although RNA circularization was first documented in the 1990s, the extent to which it occurs was not known until recent advances in high-throughput sequencing enabled the widespread identification of circular RNAs (circRNAs). Despite this, many aspects of circRNA biogenesis, structure, and function yet remain obscure. This review focuses on circular exonic RNAs, a subclass of circRNAs that are generated through backsplicing. Here, I hypothesize that RNA secondary structure can be the common factor that promotes both exon skipping and spliceosomal RNA circularization, and that backsplicing of double-stranded regions could generate topologically linked circRNA molecules. CircRNAs manifest themselves by the presence of tail-to-head exon junctions, which were previously attributed to post-transcriptional exon permutation and repetition. I revisit these observations and argue that backsplicing does not automatically imply RNA circularization because tail-to-head exon junctions give only local information about transcript architecture and, therefore, they are in principle insufficient to determine globally circular topology. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

Integrative transcriptomic analysis suggests new autoregulatory splicing events coupled with nonsense-mediated mRNA decay.

Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic analysis integrating three data sources, eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such autoregulatory feedback loops. We show that RBPs frequently bind their own pre-mRNAs, their exons respond prominently to NMD pathway disruption, and that the responding exons are enriched with nearby eCLIP peaks. We confirm previously proposed models of autoregulation in SRSF7 and U2AF1 genes and present two novel models, in which (i) SFPQ binds its mRNA and promotes switching to an alternative distal 3'-UTR that is targeted by NMD, and (ii) RPS3 binding activates a poison 5'-splice site in its pre-mRNA that leads to a frame shift and degradation by NMD. We also suggest specific splicing events that could be implicated in autoregulatory feedback loops in RBM39, HNRNPM, and U2AF2 genes. The results are available through a UCSC Genome Browser track hub.

An Evolutionary Mechanism for the Generation of Competing RNA Structures Associated with Mutually Exclusive Exons.

Alternative splicing is a commonly-used mechanism of diversifying gene products. Mutually exclusive exons (MXE) represent a particular type of alternative splicing, in which one and only one exon from an array is included in the mature RNA. A number of genes with MXE do so by using a mechanism that depends on RNA structure. Transcripts of these genes contain multiple sites called selector sequences that are all complementary to a regulatory element called the docking site; only one of the competing base pairings can form at a time, which exposes one exon from the cluster to the spliceosome. MXE tend to have similar lengths and sequence content and are believed to originate through tandem genomic duplications. Here, we report that pre-mRNAs of this class of exons have an increased capacity to fold into competing secondary structures. We propose an evolutionary mechanism for the generation of such structures via duplications that affect not only exons, but also their adjacent introns with stem-loop structures. If one of the two arms of a stem-loop is duplicated, it will generate two selector sequences that compete for the same docking site, a pattern that is associated with MXE splicing. A similar partial duplication of two independent stem-loops produces a pattern that is consistent with the so-called bidirectional pairing model. These models explain why tandem exon duplications frequently result in mutually exclusive splicing.

Towards Long-Range RNA Structure Prediction in Eukaryotic Genes.

The ability to form an intramolecular structure plays a fundamental role in eukaryotic RNA biogenesis. Proximate regions in the primary transcripts fold into a local secondary structure, which is then hierarchically assembled into a tertiary structure that is stabilized by RNA-binding proteins and long-range intramolecular base pairings. While the local RNA structure can be predicted reasonably well for short sequences, long-range structure at the scale of eukaryotic genes remains problematic from the computational standpoint. The aim of this review is to list functional examples of long-range RNA structures, to summarize current comparative methods of structure prediction, and to highlight their advances and limitations in the context of long-range RNA structures. Most comparative methods implement the “first-align-then-fold” principle, i.e., they operate on multiple sequence alignments, while functional RNA structures often reside in non-conserved parts of the primary transcripts. The opposite “first-fold-then-align” approach is currently explored to a much lesser extent. Developing novel methods in both directions will improve the performance of comparative RNA structure analysis and help discover novel long-range structures, their higher-order organization, and RNA⁻RNA interactions across the transcriptome.

The effects of death and post-mortem cold ischemia on human tissue transcriptomes.

Post-mortem tissues samples are a key resource for investigating patterns of gene expression. However, the processes triggered by death and the post-mortem interval (PMI) can significantly alter physiologically normal RNA levels. We investigate the impact of PMI on gene expression using data from multiple tissues of post-mortem donors obtained from the GTEx project. We find that many genes change expression over relatively short PMIs in a tissue-specific manner, but this potentially confounding effect in a biological analysis can be minimized by taking into account appropriate covariates. By comparing ante- and post-mortem blood samples, we identify the cascade of transcriptional events triggered by death of the organism. These events do not appear to simply reflect stochastic variation resulting from mRNA degradation, but active and ongoing regulation of transcription. Finally, we develop a model to predict the time since death from the analysis of the transcriptome of a few readily accessible tissues.

Hypoxic renal injury in newborns with abdominal compartment syndrome (clinical and experimental study).

BackgroundSurgical treatment for gastroschisis and congenital diaphragmatic hernia (CDH) commonly leads to abdominal compartment syndrome (ACS) associated with hypoxic renal injury. We hypothesized that measurement of urinary and serum concentrations of vascular endothelial growth factor (VEGF), π-glutathione S-transferase (π-GST), and monocyte chemoattractant protein-1 (MCP-1) may serve for noninvasive detection of hypoxic renal injury in such patients.MethodsIntra-abdominal pressure (IAP), renal excretory function, and the biomarker levels were analyzed before, 4, and 10 days after surgery. Association between the biomarker levels and renal histology was investigated using an original model of ACS in newborn rats.ResultsFour days after surgery, IAP increased, renal excretory function decreased, and the levels of VEGF, π-GST, and MCP-1 increased, indicating renal injury. Ten days after surgery, IAP partially decreased, renal excretory function completely restored, but the biomarker levels remained elevated, suggesting the ongoing kidney injury. In the model of ACS, increase in the biomarker levels was associated with progressing kidney morphological alteration.ConclusionSurgical treatment for gastroschisis and CDH is associated with prolonged hypoxic kidney injury despite complete restoration of renal excretory function. Follow-up measurement of VEGF, π-GST, and MCP-1 levels may provide a better tool for noninvasive assessment of renal parenchyma in newborns with ACS.