Abstract: BACKGROUND Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Abstract: TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

Abstract: Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

Abstract: Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

Abstract: In order to survive and propagate, RNA viruses must achieve a balance between the capacity for adaptation to new environmental conditions or host cells with the need to maintain an intact and replication competent genome Several virus families in the order Nidovirales, such as the coronaviruses (CoVs) must achieve these objectives with the largest and most complex replicating RNA genomes known, up to 32 kb of positive-sense RNA The CoVs encode sixteen nonstructural proteins (nsp 1-16) with known or predicted RNA synthesis and modification activities, and it has been proposed that they are also responsible for the evolution of large genomes The CoVs, including murine hepatitis virus (MHV) and SARS-CoV, encode a 3'-to-5' exoribonuclease activity (ExoN) in nsp14 Genetic inactivation of ExoN activity in engineered SARS-CoV and MHV genomes by alanine substitution at conserved DE-D-D active site residues results in viable mutants that demonstrate 15- to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other RNA viruses Thus nsp14-ExoN is essential for replication fidelity, and likely serves either as a direct mediator or regulator of a more complex RNA proofreading machine, a process previously unprecedented in RNA virus biology Elucidation of the mechanisms of nsp14-mediated proofreading will have major implications for our understanding of the evolution of RNA viruses, and also will provide a robust model to investigate the balance between fidelity, diversity and pathogenesis The discovery of a protein distinct from a viral RdRp that regulates replication fidelity also raises the possibility that RNA genome replication fidelity may be adaptable to differing replication environments and selective pressures, rather than being a fixed determinant

Abstract: Group II introns are mobile ribozymes that self-splice from precursor RNAs to yield excised intron lariat RNAs, which then invade new genomic DNA sites by reverse splicing. The introns encode a reverse transcriptase that stabilizes the catalytically active RNA structure for forward and reverse splicing, and afterwards converts the integrated intron RNA back into DNA. The characteristics of group II introns suggest that they or their close relatives were evolutionary ancestors of spliceosomal introns, the spliceosome, and retrotransposons in eukaryotes. Further, their ribozyme-based DNA integration mechanism enabled the development of group II introns into gene targeting vectors ("targetrons"), which have the unique feature of readily programmable DNA target specificity.

Abstract: Transcriptome sequencing allows for analysis of mature RNAs at base pair resolution. Here we show that RNA-seq can also be used for studying nascent RNAs undergoing transcription. We sequenced total RNA from human brain and liver and found a large fraction of reads (up to 40%) within introns. Intronic RNAs were abundant in brain tissue, particularly for genes involved in axonal growth and synaptic transmission. Moreover, we detected significant differences in intronic RNA levels between fetal and adult brains. We show that the pattern of intronic sequence read coverage is explained by nascent transcription in combination with co-transcriptional splicing. Further analysis of co-transcriptional splicing indicates a correlation between slowly removed introns and alternative splicing. Our data show that sequencing of total RNA provides unique insight into the transcriptional processes in the cell, with particular importance for normal brain development.

Abstract: To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. Eighty-seven percent of the introns assayed manifest >50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly or slowly, with ∼3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns, and introns annotated as alternative. Finally, S2 cells expressing the slow RpII215(C4) mutant show substantially less intron retention than wild-type S2 cells.

Abstract: We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

Abstract: Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.

Abstract: Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.

Abstract: Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.

Abstract: The tumor suppressor gene TP53, encoding p53, is expressed asseveral transcripts. The fully spliced p53 (FSp53) transcript enco-desthecanonicalp53protein.The alternativelysplicedp53I2 tran-script retains intron 2 andencodesD40p53 (or DNp53), an isoformlacking first 39 N-terminal residues corresponding to the maintransactivation domain. We demonstrate the formation of G-quad-ruplex structures (G4) in a GC-rich region of intron 3 that modu-lates the splicing ofintron 2. First, we show the formation of G4 insynthetic RNAs encompassing intron 3 sequences by ultravioletmelting, thermal difference spectra and circular dichroism spec-troscopy. These observations are confirmed by detection of G4-in-duced reverse transcriptase elongation stops in synthetic RNA ofintron 3. In this region, p53 pre-messenger RNA (mRNA) containsa succession of short exons (exons 2 and 3) and introns (introns 2and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by #30% theexcision ofintron 2 in a green fluorescent protein-reporter splicingassay. Moreover, treatment of lymphoblastoid cells with 360A,a synthetic ligand that binds to single-strand G4 structures, in-creasestheformation ofFSp53mRNAanddecreasesp53I2mRNAexpression. These results indicate that G4 structures in intron 3regulate the splicing of intron 2, leading to differential expressionof transcripts encoding distinct p53 isoforms.IntroductionThe tumor suppressor p53 protein controls antiproliferative responsesto various forms of stress (1). Its function is impaired in .50% ofhuman cancer, mainly by mutation (2). TP53 gene expression iscomplex, with different transcripts encoding isoforms carrying dis-tinctN- andC-termini (3,4).Todate,10isoformshavebeenidentifiedresulting from the usage of alternative promoters, splice sites and/ortranslational initiation sites (5). Several of these isoforms differ intheir N-terminal region. The N-terminus of p53 contains the maintransactivation domain (residues 1–42, transactivation domain I) aswell as the binding site of Hdm2, which targets p53 for proteasomedegradation and regulates p53 stability (1). Transcription of p53 mes-senger RNA (mRNA) from the proximal promoter generates twoproteins with distinct N-terminal domain. The first corresponds tothe canonical p53 protein, assembled from the fully spliced p53(FSp53) mRNA that retains 11 exons. This protein induces p53-me-diated growth suppression in response to stress. The second isoform,D40p53 (or DNp53), is assembled from an alternatively splicedmRNA retaining intron 2 (p53I2) and lacks the first 39 residues,corresponding to transactivation domain I, as well as Hdm2-bindingsite (3,6). The use of an internal promoter located in a region betweenintron 1 and exon 5 generates a third N-terminal isoform, D133p53,which lacks the first 132 residues (5).When expressed in excess to p53, D40p53 inhibits transcriptionalactivity and interferes in the control of cell cycle progression and apo-ptosisbyexertinganegativeeffectontheexpressionofp53-targetgenes(3,6,7). However, the biological circumstances and the molecularmechanisms regulating D40p53 expression are still poorly known. Re-tentionofintron2inp53I2mRNAintroducesseveralstopcodonsinthereading frame of AUG 1, thus preventing the synthesis of a full-lengthp53protein.However,p53I2mRNAcanbetranslatedusingAUG40asinitiation site, generating a protein isoform which differs from thecanonical p53 by the lack of the first 39 residues. Expression ofp53I2 transcript has been reported in cell lines, such as MCF-7, innormallymphocytesandinprimarymelanomaisolates(7,8).However,themechanismthatregulatesthesplicingofp53pre-mRNAintoFSp53or p53I2 is not understood. D40p53 protein isoform can also be pro-duced by internal ribosomal entry site-regulated internal initiation oftranslation using FSp53 mRNA (9,10).In recent years, it has been proposed that tridimensional RNA struc-tures such as G-quadruplexes may play important roles in regulatingsplicing (11). These structures result of the propensity of G-rich se-quences to fold into four-stranded cation-dependent structures (12).They are formed by the interaction of four guanines organized in acyclic Hoogsteen hydrogen bonding arrangement termed a G-quartetand by the stacking of several G-quartets (Figure 1A). It is estimatedthatover376 000sequencesinthehumangenomehavethepotentialtoadopt G-quadruplex structures, most of them located in non-codingregions (13,14).Atthe RNA level, G-quadruplexes may play a numberof roles.Innon-coding RNAs,they can affecttheir structuresand func-tions (15,16). In 5#-untranslated region of mRNAs, G-quadruplexeshave been shown to modulate translation (17,18). When present inintrons, G-quadruplexes can affect the splicing and expression patternsof genessuchashTERT (humantelomerasereversetranscriptase), Bcl-xL or FMRP (Fragile X mental retardation protein) (11,19,20).The sequence of intron 3 in TP53 contains tracts of G bases orga-nized in a pattern similar to the one of regions forming G-quadruplexstructures. Since exon 3 in TP53 is extremely short (22 bp), we rea-soned that motifs located in intron 3 might have an effect on theregulation of the splicing of intron 2. In this study, we provide anevidence for the formation of G-quadruplex in TP53 intron 3 and thatthese G-quadruplex structures may affect the splicing of intron 2,modulating the synthesis of either FSp53 (intron 2 spliced out) orp53I2 (intron 2 retained) mRNAs, which encode different p53 proteinisoforms.Materials and methods

Abstract: The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.

Abstract: Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK–associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

Abstract: It has been widely acknowledged that non-coding RNAs are master-regulators of genomic functions. However, the significance of the presence of ncRNA within introns has not received proper attention. ncRNA within introns are commonly produced through the post-splicing process and are specific signals of gene transcription events, impacting many other genes and modulating their expression. This study, along with the following discussion, details the association of thousands of ncRNAs—snoRNA, miRNA, siRNA, piRNA and long ncRNA—within human introns. We propose that such an association between human introns and ncRNAs has a pronounced synergistic effect with important implications for fine-tuning gene expression patterns across the entire genome.

Abstract: The β-amyloid peptide that accumulate in Alzheimer's disease (AD) brain derive from proteolytic processing of the amyloid precursor protein (APP). Recent evidence suggest that microRNAs (miRNAs) participate in the post-transcriptional regulation of APP expression. Because gene dosage effects of the APP gene can cause genetic AD, dysregulation of the miRNA network could contribute significantly to disease. Here, we present evidence that, besides APP expression regulation, miRNAs are equally involved in the regulation of neuronal APP mRNA alternative splicing. Lack of miRNAs in post-mitotic neurons in vivo is associated with APP exons 7 and 8 inclusion, while ectopic expression of miR-124, an abundant neuronal-specific miRNA, reversed these effects in cultured neurons. Similar results were obtained by depletion of endogenous polypyrimidine tract binding protein 1 (PTBP1) in cells, a recognized miR-124 target gene. Furthermore, PTBP1 levels correlate with the presence of APP exons 7 and 8, while PTBP2 levels correlate with the skipping of these exons during neuronal differentiation. Finally, we show that miR-124 is down-regulated in AD brain. In sum, our results suggest that specific miRNAs are involved in the fine-tuning of APP alternative splicing in neurons. Since abnormal neuronal splicing of APP affects β-amyloid peptide production, these results could contribute to the understanding of the implication of miRNAs in brain health and disease.

Abstract: Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.

Abstract: Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Abstract: Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

Abstract: Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines. Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4, may play functional roles in cancer.

Abstract: Signal transducer and activator of transcription 3 (STAT3) plays a central role in the activation of multiple oncogenic pathways. Splicing variant STAT3β uses an alternative acceptor site within exon 23 that leads to a truncated isoform lacking the C-terminal transactivation domain. Depending on the context, STAT3β can act as a dominant-negative regulator of transcription and promote apoptosis. We show that modified antisense oligonucleotides targeted to a splicing enhancer that regulates STAT3 exon 23 alternative splicing specifically promote a shift of expression from STAT3α to STAT3β. Induction of endogenous STAT3β leads to apoptosis and cell-cycle arrest in cell lines with persistent STAT3 tyrosine phosphorylation compared with total STAT3 knockdown obtained by forced splicing-dependent nonsense-mediated decay (FSD-NMD). Comparison of the molecular effects of splicing redirection to STAT3 knockdown reveals a unique STAT3β signature, with a down-regulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1β) distinct from canonical STAT3 targets typically associated with total STAT3 knockdown. Furthermore, similar in vivo redirection of STAT3 alternative splicing leads to tumor regression in a xenograft cancer model, demonstrating how pharmacological manipulation of a single key splicing event can manifest powerful antitumorigenic properties and validating endogenous splicing reprogramming as an effective cancer therapeutic approach.

Abstract: In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death‐domain adaptor protein Insuloma‐Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti‐apoptotic isoform (MAP‐kinase activating death domain protein, MADD), which effectively redirects TNF‐α/TRAIL‐induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand‐independent, constitutively active Recepteur d′Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16‐ or RON exon 11‐containing variants, respectively, using isoform‐specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness.

Abstract: RNA-seq was used to generate an extensive map of the Drosophila melanogaster transcriptome by broad sampling of 10 developmental stages. In total, 142.2 million uniquely mapped 64–100-bp paired-end reads were generated on the Illumina GA II yielding 356× sequencing coverage. More than 95% of FlyBase genes and 90% of splicing junctions were observed. Modifications to 30% of FlyBase gene models were made by extension of untranslated regions, inclusion of novel exons, and identification of novel splicing events. A total of 319 novel transcripts were identified, representing a 2% increase over the current annotation. Alternate splicing was observed in 31% of D. melanogaster genes, a 38% increase over previous estimations, but significantly less than that observed in higher organisms. Much of this splicing is subtle such as tandem alternate splice sites.

Abstract: Alternative splicing (AS) is a widespread mechanism underlying the generation of proteomic and regulatory complexity. However, which of the myriad of human AS events play important roles in disease is largely unknown. To identify frequently occurring AS events in lung cancer, we used AS microarray profiling and reverse transcription-PCR (RT-PCR) assays to survey patient-matched normal and adenocarcinoma tumor tissues from the lungs of 29 individuals diagnosed with non-small cell lung cancer (NSCLC). Of 5,183 profiled alternative exons, four displayed tumor-associated changes in the majority of the patients. These events affected transcripts from the VEGFA, MACF1, APP, and NUMB genes. Similar AS changes were detected in NUMB and APP transcripts in primary breast and colon tumors. Tumor-associated increases in NUMB exon 9 inclusion correlated with reduced levels of NUMB protein expression and activation of the Notch signaling pathway, an event that has been linked to tumorigenesis. Moreover, short hairpin RNA (shRNA) knockdown of NUMB followed by isoform-specific rescue revealed that expression of the exon 9-skipped (nontumor) isoform represses Notch target gene activation whereas expression of the exon 9-included (tumor) isoform lacks this activity and is capable of promoting cell proliferation. The results thus reveal widespread AS changes in NSCLC that impact cell signaling in a manner that likely contributes to tumorigenesis.