Autoregulation of RBM10 and cross-regulation of RBM10/RBM5 via alternative splicing-coupled nonsense-mediated decay.

Mutations in the spliceosomal RNA binding protein RBM10 cause TARP syndrome and are frequently observed in lung adenocarcinoma (LUAD). We have previously shown that RBM10 enhances exon skipping of its target genes, including its paralog RBM5. Here, we report that RBM10 negatively regulates its own mRNA and protein expression and that of RBM5 by promoting alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). Through computational analysis and experimental validation, we identified RBM10-promoted skipping of exon 6 or 12 in RBM10 and exon 6 or 16 in RBM5 as the underlying AS-NMD events. Importantly, we showed that LUAD-associated mutations affecting splice sites of RBM10 exons 6 or 12 abolished exon inclusion and correlated with reduced expression of RBM10 RNA. Together, our investigations have revealed novel molecular mechanisms underlying RBM10 autoregulation and cross-regulation of RBM5, thereby providing insights concerning the functions of RBM10 under various physiological and pathological conditions. Our combined computational and experimental approach should be useful for elucidating the role of AS-NMD in auto- and cross-regulation by other splicing regulators.

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing.

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18,496 isoforms, out of 19,008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons.

Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth.

RATIONALE: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. OBJECTIVE: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). METHODS AND RESULTS: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. CONCLUSIONS: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.

Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3.

OBJECTIVE: Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. APPROACH AND RESULTS: Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2 represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2-mediated reduction in angiogenic sprouting and network formation. CONCLUSIONS: Our data demonstrate that shear stress-mediated repression of endothelial cell metabolism via KLF2 and PFKFB3 controls endothelial cell phenotype.

Argonaute2 regulates the pancreatic ß-cell secretome.

Argonaute2 (Ago2) is an established component of the microRNA-induced silencing complex. Similar to miR-375 loss-of-function studies, inhibition of Ago2 in the pancreatic ß-cell resulted in enhanced insulin release underlining the relationship between these two genes. Moreover, as the most abundant microRNA in pancreatic endocrine cells, miR-375 was also observed to be enriched in Ago2-associated complexes. Both Ago2 and miR-375 regulate the pancreatic ß-cell secretome, and by using quantitative mass spectrometry, we identified the enhanced release of a set of proteins or secretion "signatures " in response to a glucose stimulus using the murine ß-cell line MIN6. In addition, the loss of Ago2 resulted in the increased expression of miR-375 target genes, including gephyrin and ywhaz. These targets positively contribute to exocytosis indicating they may mediate the functional role of both miR-375 and Ago proteins in the pancreatic ß-cell by influencing the secretory pathway. This study specifically addresses the role of Ago2 in the systemic release of proteins from ß-cells and highlights the contribution of the microRNA pathway to the function of this cell type.

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery.

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5' tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.

De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics.

Freshwater planaria are a very attractive model system for stem cell biology, tissue homeostasis, and regeneration. The genome of the planarian Schmidtea mediterranea has recently been sequenced and is estimated to contain >20,000 protein-encoding genes. However, the characterization of its transcriptome is far from complete. Furthermore, not a single proteome of the entire phylum has been assayed on a genome-wide level. We devised an efficient sequencing strategy that allowed us to de novo assemble a major fraction of the S. mediterranea transcriptome. We then used independent assays and massive shotgun proteomics to validate the authenticity of transcripts. In total, our de novo assembly yielded 18,619 candidate transcripts with a mean length of 1118 nt after filtering. A total of 17,564 candidate transcripts could be mapped to 15,284 distinct loci on the current genome reference sequence. RACE confirmed complete or almost complete 5' and 3' ends for 22/24 transcripts. The frequencies of frame shifts, fusion, and fission events in the assembled transcripts were computationally estimated to be 4.2%-13%, 0%-3.7%, and 2.6%, respectively. Our shotgun proteomics produced 16,135 distinct peptides that validated 4200 transcripts (FDR ≤1%). The catalog of transcripts assembled in this study, together with the identified peptides, dramatically expands and refines planarian gene annotation, demonstrated by validation of several previously unknown transcripts with stem cell-dependent expression patterns. In addition, our robust transcriptome characterization pipeline could be applied to other organisms without genome assembly. All of our data, including homology annotation, are freely available at SmedGD, the S. mediterranea genome database.

A systematic evaluation of hybridization-based mouse exome capture system.

BACKGROUND: Exome sequencing is increasingly used to search for phenotypically-relevant sequence variants in the mouse genome. All of the current hybridization-based mouse exome capture systems are designed based on the genome reference sequences of the C57BL/6 J strain. Given that the substantial sequence divergence exists between C57BL/6 J and other distantly-related strains, the impact of sequence divergence on the efficiency of such capture systems needs to be systematically evaluated before they can be widely applied to the study of those strains. RESULTS: Using the Agilent SureSelect mouse exome capture system, we performed exome sequencing on F1 generation hybrid mice that were derived by crossing two divergent strains, C57BL/6 J and SPRET/EiJ. Our results showed that the C57BL/6 J-based probes captured the sequences derived from C57BL/6 J alleles more efficiently and that the bias was higher for the target regions with greater sequence divergence. At low sequencing depths, the bias also affected the efficiency of variant detection. However, the effects became negligible when sufficient sequencing depth was achieved. CONCLUSION: Sufficient sequence depth needs to be planned to match the sequence divergence between C57BL/6 J and the strain to be studied, when the C57BL/6 J-based Agilent SureSelect exome capture system is to be used.

Corrigendum: A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

[This corrects the article DOI: 10.3389/fnmol.2017.00439.].

Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus.

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients.

Acfs: accurate circRNA identification and quantification from RNA-Seq data.

Circular RNAs (circRNAs) are a group of single-stranded RNAs in closed circular form. They are splicing-generated, widely expressed in various tissues and have functional implications in development and diseases. To facilitate genome-wide characterization of circRNAs using RNA-Seq data, we present a freely available software package named acfs. Acfs allows de novo, accurate and fast identification and abundance quantification of circRNAs from single- and paired-ended RNA-Seq data. On simulated datasets, acfs achieved the highest F1 accuracy and lowest false discovery rate among current state-of-the-art tools. On real-world datasets, acfs efficiently identified more bona fide circRNAs. Furthermore, we demonstrated the power of circRNA analysis on two leukemia datasets. We identified a set of circRNAs that are differentially expressed between AML and APL samples, which might shed light on the potential molecular classification of complex diseases using circRNA profiles. Moreover, chromosomal translocation, as manifested in numerous diseases, could produce not only fusion transcripts but also fusion circRNAs of clinical relevance. Featured with high accuracy, low FDR and the ability to identify fusion circRNAs, we believe that acfs is well suited for a wide spectrum of applications in characterizing the landscape of circRNAs from non-model organisms to cancer biology.

Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated post-transcriptional regulation.

Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3' untranslated region (3' UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stem-loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3' UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.

Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure.

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.

Argonaute2 mediates compensatory expansion of the pancreatic ß cell.

Pancreatic ß cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in ß cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory ß cell expansion. Loss of Ago2 during insulin resistance blocked ß cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and ß cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.