Search

RNA editing, the endogenous modification of nucleic acids, is known to be altered in genes with important neurological function in schizophrenia (SCZ). However, the global profile and molecular functions of disease-associated RNA editing remain unclear. Here, we analyzed RNA editing in postmortem brains of four SCZ cohorts and uncovered a significant and reproducible trend of hypoediting in patients of European descent. We report a set of SCZ-associated editing sites via WGCNA analysis, shared across cohorts. Using massively parallel reporter assays and bioinformatic analyses, we observed that differential 3′ untranslated region (3′UTR) editing sites affecting host gene expression were enriched for mitochondrial processes. Furthermore, we characterized the impact of two recoding sites in the mitofusin 1 ( MFN1 ) gene and showed their functional relevance to mitochondrial fusion and cellular apoptosis. Our study reveals a global reduction of editing in SCZ and a compelling link between editing and mitochondrial function in the disease.

Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1-deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1-/- liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.

Motivation Alternative polyadenylation (polyA) sites near the 3′ end of a pre-mRNA create multiple mRNA transcripts with different 3′ untranslated regions (3′ UTRs). The sequence elements of a 3′ UTR are essential for many biological activities such as mRNA stability, sub-cellular localization, protein translation, protein binding and translation efficiency. Moreover, numerous studies in the literature have reported the correlation between diseases and the shortening (or lengthening) of 3′ UTRs. As alternative polyA sites are common in mammalian genes, several machine learning tools have been published for predicting polyA sites from sequence data. These tools either consider limited sequence features or use relatively old algorithms for polyA site prediction. Moreover, none of the previous tools consider RNA secondary structures as a feature to predict polyA sites. Results In this paper, we propose a new deep learning model, called DeepPASTA, for predicting polyA sites from both sequence and RNA secondary structure data. The model is then extended to predict tissue-specific polyA sites. Moreover, the tool can predict the most dominant (i.e. frequently used) polyA site of a gene in a specific tissue and relative dominance when two polyA sites of the same gene are given. Our extensive experiments demonstrate that DeepPASTA signisficantly outperforms the existing tools for polyA site prediction and tissue-specific relative and absolute dominant polyA site prediction. Availability and implementation https://github.com/arefeen/DeepPASTA Supplementary information Supplementary data are available at Bioinformatics online.

BackgroundRNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance.ResultsHere, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response.ConclusionsOur study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.

Motivation RNA-sequencing (RNA-seq) enables global identification of RNA-editing sites in biological systems and disease. A salient step in many studies is to identify editing sites that statistically associate with treatment (e.g. case versus control) or covary with biological factors, such as age. However, RNA-seq has technical features that incumbent tests (e.g. t-test and linear regression) do not consider, which can lead to false positives and false negatives. Results In this study, we demonstrate the limitations of currently used tests and introduce the method, RNA-editing tests (REDITs), a suite of tests that employ beta-binomial models to identify differential RNA editing. The tests in REDITs have higher sensitivity than other tests, while also maintaining the type I error (false positive) rate at the nominal level. Applied to the GTEx dataset, we unveil RNA-editing changes associated with age and gender, and differential recoding profiles between brain regions. Availability and implementation REDITs are implemented as functions in R and freely available for download at https://github.com/gxiaolab/REDITs. The repository also provides a code example for leveraging parallelization using multiple cores.

microRNAs (miRNAs) are small non-coding RNAs that play critical roles in gene regulation. The presence of miRNAs in extracellular biofluids is increasingly recognized. However, most previous characterization of extracellular miRNAs focused on their overall expression levels. Alternative sequence isoforms and modifications of miRNAs were rarely considered in the extracellular space. Here, we developed a highly accurate bioinformatic method, called miNTA, to identify 3’ non-templated additions (NTAs) of miRNAs using small RNA-sequencing data. Using miNTA, we conducted an in-depth analysis of miRNA 3’ NTA profiles in 1047 extracellular RNA-sequencing data sets of 4 types of biofluids. This analysis identified hundreds of miRNAs with 3’ uridylation or adenylation, with the former being more prevalent. Among these miRNAs, up to 53% (22%) had an average 3’ uridylation (adenylation) level of at least 10% in a specific biofluid. Strikingly, we found that 3’ uridylation levels enabled segregation of different types of biofluids, more effectively than overall miRNA expression levels. This observation suggests that 3’ NTA levels possess fluid-specific information relatively robust to batch effects. In addition, we observed that extracellular miRNAs with 3’ uridylations are enriched in processes related to angiogenesis, apoptosis, and inflammatory response, and this type of modification may stabilize base-pairing between miRNAs and their target genes. Together, our study provides a comprehensive landscape of miRNA NTAs in human biofluids, which paves way for further biomarker discoveries. The insights generated in our work built a foundation for future functional, mechanistic, and translational discoveries.

Recent studies revealed global shifts in RNA editing, the modification of RNA sequences, across many cancers. Besides a few sites implicated in tumorigenesis or metastasis, most tumor-associated sites, predominantly in noncoding regions, have unknown function. Here, we characterize editing profiles between epithelial (E) and mesenchymal (M) phenotypes in seven cancer types, as epithelial-mesenchymal transition (EMT) is a key paradigm for metastasis. We observe distinct editing patterns between E and M tumors and EMT induction upon loss of ADAR enzymes in cultured cells. E-M differential sites are highly enriched in genes involved in immune and viral processes, some of which regulate mRNA abundance of their respective genes. We identify a novel mechanism in which ILF3 preferentially stabilizes edited transcripts. Among editing-dependent ILF3 targets is the transcript encoding PKR, a crucial player in immune response. Our study demonstrates the broad impact of RNA editing in cancer and relevance of editing to cancer-related immune pathways.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH’s effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

BACKGROUND It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649–6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482–696 microRNAs (miRNAs) and 190–214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

Alternative transcription start (ATS) and alternative polyadenylation (APA) create alternative RNA isoforms and modulate many aspects of RNA expression and protein production. However, ATS and APA remain difficult to detect in RNA sequencing (RNA-seq). Here, we developed mountainClimber, a de novo cumulative-sum-based approach to identify ATS and APA as change points. Unlike many existing methods, mountainClimber runs on a single sample and identifies multiple ATS or APA sites anywhere in the transcript. We analyzed 2,342 GTEx samples (36 tissues, 215 individuals) and found that tissue type is the predominant driver of transcript end variations. 75% and 65% of genes exhibited differential APA and ATS across tissues, respectively. In particular, testis displayed longer 5' untranslated regions (UTRs) and shorter 3' UTRs, often in genes related to testis-specific biology. Overall, we report the largest study of transcript ends across human tissues to our knowledge. mountainClimber is available at github.com/gxiaolab/mountainClimber.

RNA editing introduces single nucleotide changes to RNA, thus potentially diversifying gene expression. Recent studies have reported significant changes in RNA editing profiles in disease and development. The functional consequences of these widespread alterations remain elusive because of the unknown function of most RNA editing sites. Here, we carry out a comprehensive analysis of A-to-I editomes in human populations. Surprisingly, we observe highly similar editing profiles across populations despite striking differences in the expression levels of ADAR genes. Striving to explain this discrepancy, we uncover a functional mechanism of A-to-I editing in regulating mRNA abundance. We show that A-to-I editing stabilizes RNA secondary structures and reduces the accessibility of AGO2-miRNA to target sites in mRNAs. The editing-dependent stabilization of mRNAs in turn alters the observed editing levels in the stable RNA repertoire. Our study provides valuable insights into the functional impact of RNA editing in human cells., RNA A-to-I editing introduces single nucleotide changes to RNA, but its role in cells remains unclear. Here, the authors analyse A-to-I editomes in human samples and find that A-to-I editing stabilizes RNA secondary structures and reduces the accessibility of AGO2-miRNA to target sites in mRNAs.

© 2019 Elsevier Inc. The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data. The Extracellular RNA Communication Consortium (ERCC) presents progress toward understanding the biology of extracellular RNAs and their use as biomarkers and therapeutics.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs., Differential binding of RNA-binding proteins mediated by genetic variants (GVs) can influence posttranscriptional regulation. Here, the authors develop BEAPR, a computational approach to identify allele-specific binding events in eCLIP-Seq data.

Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing., Giovanni Quinones-Valdez et al. examined the role of over 200 RNA-binding proteins in mediating A-to-I RNA editing. They identified several RNA-binding proteins that regulate ADAR1 expression, interaction, or binding with Alu elements in a cell type-specific manner.

Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting that cis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon.

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.

Human speech is one of the few examples of vocal learning among mammals yet ~half of avian species exhibit this ability. Its neurogenetic basis is largely unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. We manipulated FoxP2 isoforms in Area X, a song-specific region of the avian striatopallidum analogous to human anterior striatum, during a critical period for song development. We delineate, for the first time, unique contributions of each isoform to vocal learning. Weighted gene coexpression network analysis of RNA-seq data revealed gene modules correlated to singing, learning, or vocal variability. Coexpression related to singing was found in juvenile and adult Area X whereas coexpression correlated to learning was unique to juveniles. The confluence of learning and singing coexpression in juvenile Area X may underscore molecular processes that drive vocal learning in young zebra finches and, by analogy, humans., eLife digest Songbirds, much like in humans, have a critical period in youth when they are best at learning vocal communication skills. In birds, this is when they learn a song they will use later in life as a courtship song. In humans, this is when language skills are most easily learned. After this critical period ends, it is much harder for people to learn languages, and for certain bird species to learn their song. When birds sing every morning, the activity of a gene called FoxP2 drops, which causes a coordinated change in the activity of thousands of other genes. It is suspected that FoxP2 – and the changes it causes – could be a part of the molecular basis for vocal learning. FoxP2 is also known to play a role in speech in humans, and both birds and humans have a long and a short version of this gene. Previous research has shown that when the long version of the gene was altered so its activity would no longer decrease when birds were singing, the birds failed to learn their song. Moreover, humans with a mutation in the long version have problems with their speech. However, until now, it was not known if modifications to the short version had the same effect. Burkett et al. investigated whether there was a noticeable pattern in the effects of FoxP2 before and after the critical period in a songbird. The analysis found that during the critical period, a set of genes changed together as young birds learned to sing. This particular pattern disappeared as the birds aged and the critical period ended. Burkett et al. confirmed that when birds had the long version of FoxP2 altered, they were less able to learn. However, changing the short version of FoxP2 had little effect on learning but led to changes in the birds’ song. The genetic pathways identified in the experiments are known to be present in many different species, including humans. Related pathways have also been found to play a role in non-vocal learning in organisms as distantly related as rats and snails. This suggests that they could be acting as a blueprint for learning new skills. Few treatments for language impairments have been developed so far due to poor understanding of the molecular basis for vocal communication. The findings of this study could help to create new treatments for speech problems in people, such as children with autism or people with mutated versions of FoxP2.

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

Motivation Analysis of RNA sequencing (RNA-Seq) data in human saliva is challenging. Lack of standardization and unification of the bioinformatic procedures undermines saliva‘s diagnostic potential. Thus, it motivated us to perform this study. Results We applied principal pipelines for bioinformatic analysis of small RNA-Seq data of saliva of 98 healthy Korean volunteers including either direct or indirect mapping of the reads to the human genome using Bowtie1. Analysis of alignments to exogenous genomes by another pipeline revealed that almost all of the reads map to bacterial genomes. Thus, salivary exRNA has fundamental properties that warrant the design of unique additional steps while performing the bioinformatic analysis. Our pipelines can serve as potential guidelines for processing of RNA-Seq data of human saliva. Availability and implementation Processing and analysis results of the experimental data generated by the exceRpt (v4.6.3) small RNA-seq pipeline (github.gersteinlab.org/exceRpt) are available from exRNA atlas (exrna-atlas.org). Alignment to exogenous genomes and their quantification results were used in this paper for the analyses of small RNAs of exogenous origin.

Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.

Background— Cardiac maturation during perinatal transition of heart is critical for functional adaptation to hemodynamic load and nutrient environment. Perturbation in this process has major implications in congenital heart defects. Transcriptome programming during perinatal stages is an important information but incomplete in current literature, particularly, the expression profiles of the long noncoding RNAs (lncRNAs) are not fully elucidated. Methods and Results— From comprehensive analysis of transcriptomes derived from neonatal mouse heart left and right ventricles, a total of 45 167 unique transcripts were identified, including 21 916 known and 2033 novel lncRNAs. Among these lncRNAs, 196 exhibited significant dynamic regulation along maturation process. By implementing parallel weighted gene co-expression network analysis of mRNA and lncRNA data sets, several lncRNA modules coordinately expressed in a developmental manner similar to protein coding genes, while few lncRNAs revealed chamber-specific patterns. Out of 2262 lncRNAs located within 50 kb of protein coding genes, 5% significantly correlate with the expression of their neighboring genes. The impact of Ppp1r1b-lncRNA on the corresponding partner gene Tcap was validated in cultured myoblasts. This concordant regulation was also conserved in human infantile hearts. Furthermore, the Ppp1r1b-lncRNA/Tcap expression ratio was identified as a molecular signature that differentiated congenital heart defect phenotypes. Conclusions— The study provides the first high-resolution landscape on neonatal cardiac lncRNAs and reveals their potential interaction with mRNA transcriptome during cardiac maturation. Ppp1r1b-lncRNA was identified as a regulator of Tcap expression, with dynamic interaction in postnatal cardiac development and congenital heart defects.

RNA-sequencing (RNA-Seq) technologies hold enormous promise for novel discoveries in genomics and transcriptomics. In the past year, a surge of reports has analyzed RNA-Seq data to gain a global view of the RNA editome. Opposing results have been presented, giving rise to extensive debate surrounding one of the first such studies in which a daunting list of all 12 types of RNA–DNA differences (RDDs) were identified. Although a consensus is forming that some of the initial “paradigm-shifting” results of this study may be questionable, recent reports on this topic differed in terms of the number and relative abundance of each type of RDD. Many outstanding issues exist, most importantly, the choice of bioinformatic approaches. Here we discuss the critical data analysis and experimental design issues of such studies to enable improved systematic investigation of the largely unexplored frontier of single-nucleotide variants in RNA.

Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation. DOI: http://dx.doi.org/10.7554/eLife.15614.001, eLife digest Metabolism is a term that describes all the chemical reactions that are involved in keeping a living organism alive. Diseases related to metabolism – such as obesity, heart disease and diabetes – are a major health problem in the Western world. The causes of these diseases are complex and include both environmental factors, such as diet and exercise, and genetics. Indeed, many genetic variants that contribute to obesity have been uncovered in both humans and mice. However, it is only dimly understood how these genetic variants affect the underlying networks of interacting genes that cause metabolic disorders. Measuring gene activity or expression, and tracing how genetic instructions are carried from DNA into RNA and proteins, can reliably identify groups of genes that correlate with metabolic traits in specific organs. This strategy was successfully used in previous studies to reveal new information about abnormalities linked to obesity in specific tissues such as the liver and fat tissues. It was also shown that this approach might suggest new molecules that could be targeted to treat metabolic disorders. A brain region called the hypothalamus is key to the control of metabolism, including feeding behavior and obesity. Hasin-Brumshtein et al. set out to explore gene expression in the hypothalamus of 99 different strains of mice, in the hope that the data will help identify new connections between gene expression and metabolism. This approach showed that thousands of new and known genes are expressed in the mouse hypothalamus, some of which coded for proteins, and some of which did not. Hasin-Brumshtein et al. uncovered two genetic variants that controlled the expression of hundreds of other genes. Further analysis then revealed thousands of genetic variants that regulated the expression of, and type of RNA (so-called "spliceforms") produced from neighboring genes. Also, the expression of many individual genes showed significant similarities with about 150 metabolic measurements that had been evaluated previously in the mice. This new dataset is a unique resource that can be coupled with different approaches to test existing ideas and develop new ones about the role of particular genes or genetic mechanisms in obesity. Future studies will likely focus on new genes that show strong associations with attributes that are relevant to metabolic disorders, such as insulin levels, weight and fat mass. DOI: http://dx.doi.org/10.7554/eLife.15614.002

In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation.

Epigenetic reprogramming is a critical process of pathological gene induction during cardiac hypertrophy and remodeling, but the underlying regulatory mechanisms remain to be elucidated. Here we identified a heart-enriched long noncoding (lnc)RNA, named cardiac-hypertrophy-associated epigenetic regulator (Chaer), which is necessary for the development of cardiac hypertrophy. Mechanistically, Chaer directly interacts with the catalytic subunit of polycomb repressor complex 2 (PRC2). This interaction, which is mediated by a 66-mer motif in Chaer, interferes with PRC2 targeting to genomic loci, thereby inhibiting histone H3 lysine 27 methylation at the promoter regions of genes involved in cardiac hypertrophy. The interaction between Chaer and PRC2 is transiently induced after hormone or stress stimulation in a process involving mammalian target of rapamycin complex 1, and this interaction is a prerequisite for epigenetic reprogramming and induction of genes involved in hypertrophy. Inhibition of Chaer expression in the heart before, but not after, the onset of pressure overload substantially attenuates cardiac hypertrophy and dysfunction. Our study reveals that stress-induced pathological gene activation in the heart requires a previously uncharacterized lncRNA-dependent epigenetic checkpoint.

Motivation: Accurate identification of genetic variants such as single-nucleotide polymorphisms (SNPs) or RNA editing sites from RNA-Seq reads is important, yet challenging, because it necessitates a very low false-positive rate in read mapping. Although many read aligners are available, no single aligner was specifically developed or tested as an effective tool for SNP and RNA editing prediction. Results: We present RASER, an accurate read aligner with novel mapping schemes and index tree structure that aims to reduce false-positive mappings due to existence of highly similar regions. We demonstrate that RASER shows the best mapping accuracy compared with other popular algorithms and highest sensitivity in identifying multiply mapped reads. As a result, RASER displays superb efficacy in unbiased mapping of the alternative alleles of SNPs and in identification of RNA editing sites. Availability and implementation: RASER is written in C++ and freely available for download at https://github.com/jaegyoonahn/RASER. Contact: gxxiao@ucla.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Pre-mRNA splicing is an essential RNA processing step in eukaryotes. Alternative splicing generates distinct spliced isoforms of the same gene, thereby dramatically increasing transcriptome diversity. Since most human genes undergo alternative splicing, this process contributes to a wide spectrum of biological functions in healthy and disease states. Splicing is closely regulated by various cis-regulatory elements and trans-factors. With the advent of high-throughput experimental technologies and bioinformatic algorithms, we now have powerful means to study alternative splicing globally and uncover its functional impact and regulatory mechanisms. As more RNA sequencing (RNA-Seq) data from normal and disease conditions are becoming available, many studies are underway to dissect global misregulation of splicing in diseases and develop novel splicing-targeted therapeutics. In this chapter, we first discuss the experimental and bioinformatic approaches for identification of alternative splicing, followed by a comprehensive review on the state-of-the-art methodologies to study splicing regulation. In addition, we discuss the current challenges and open questions in the RNA splicing field including gene expression kinetics, co-transcriptional splicing, and therapeutic approaches targeting splicing.

Rationale: Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level. Objective: To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels. Methods and Results: In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (ie, novel genes), and long noncoding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts after pressure-overload–induced by transaortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNA-Seq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations. Conclusions: Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.

Alternative splicing (AS) has emerged as a key mechanism that accounts for gene expression diversity in metazoan organisms. Splicing is tightly regulated by a repertoire of RNA and protein factors and RNA sequence elements that function in a cooperative manner. Systems-level experimental and computational approaches have been instrumental in establishing comprehensive profiles of transcript variants generated by AS. In addition, systems biology approaches are starting to define how combinatorial splicing regulation shapes the complex splicing phenotypes observed in different tissue types and developmental stages and under different conditions. Here, we review recent progress in these areas. Copyright © 2010 John Wiley & Sons, Inc. For further resources related to this article, please visit the WIREs website

BACKGROUND Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

RNA editing enhances the diversity of gene products at the post-transcriptional level. Approaches for genome-wide identification of RNA editing face two main challenges: separating true editing sites from false discoveries and accurate estimation of editing levels. We developed an approach to analyze transcriptome sequencing data (RNA-seq) for global identification of RNA editing in cells for which whole-genome sequencing data are available. We applied the method to analyze RNA-seq data of a human glioblastoma cell line, U87MG. Around 10,000 DNA–RNA differences were identified, the majority being putative A-to-I editing sites. These predicted A-to-I events were associated with a low false-discovery rate (∼5%). Moreover, the estimated editing levels from RNA-seq correlated well with those based on traditional clonal sequencing. Our results further facilitated unbiased characterization of the sequence and evolutionary features flanking predicted A-to-I editing sites and discovery of a conserved RNA structural motif that may be functionally relevant to editing. Genes with predicted A-to-I editing were significantly enriched with those known to be involved in cancer, supporting the potential importance of cancer-specific RNA editing. A similar profile of DNA–RNA differences as in U87MG was predicted for another RNA-seq data set obtained from primary breast cancer samples. Remarkably, significant overlap exists between the putative editing sites of the two transcriptomes despite their difference in cell type, cancer type, and genomic backgrounds. Our approach enabled de novo identification of the RNA editome, which sets the stage for further mechanistic studies of this important step of post-transcriptional regulation.

Accurate and efficient splicing of eukaryotic pre-mRNAs requires recognition by trans -acting factors of a complex array of cis -acting RNA elements. Here, we developed a generalized Bayesian network to model the coevolution of splicing cis elements in diverse eukaryotic taxa. Cross-exon but not cross-intron compensatory interactions between the 5′ splice site (5′ss) and 3′ splice site (3′ss) were observed in human/mouse, indicating that the exon is the primary evolutionary unit in mammals. Studied plants, fungi, and invertebrates exhibited exclusively cross-intron interactions, suggesting that intron definition drives evolution in these organisms. In mammals, 5′ss strength and the strength of several classes of exonic splicing silencers (ESSs) evolved in a correlated way, whereas specific exonic splicing enhancers (ESEs), including motifs associated with hTra2, SRp55, and SRp20, evolved in a compensatory manner relative to the 5′ss and 3′ss. Interactions between specific ESS or ESE motifs were not observed, suggesting that elements bound by different factors are not commonly interchangeable. Thus, the splicing elements defining exons coevolve in a way that preserves overall exon strength, allowing specific elements to substitute for loss or weakening of others.

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.