Showing papers on "Exon published in 2011"

The developmental transcriptome of Drosophila melanogaster

Abstract: Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Somatic SF3B1 Mutation in Myelodysplasia with Ring Sideroblasts

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.).

Characterizing the RNA targets and position-dependent splicing regulation by TDP-43

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.

CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

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.

MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas.

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.

Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity.

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

Group II Introns: Mobile Ribozymes that Invade DNA

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.

Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

Abstract: It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain

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.

Conservation of an RNA Regulatory Map Between Drosophila and Mammals

Abstract: Alternative splicing is a process by which multiple messenger RNAs (mRNAs) can be generated by joining exons together in different combinations. This process is used to both increase protein diversity and to regulate gene expression (Nilsen and Graveley 2010). Approximately 95% of human genes contain introns and therefore have the potential to be alternatively spliced. Recent deep sequencing surveys of 10 human tissues found that nearly all (95%–98%) multi-exon human genes are alternatively spliced (Pan et al. 2008; Wang et al. 2008). Given the ubiquity of alternative splicing and the key roles it plays in the control of gene expression, it is important to develop a complete understanding of the mechanisms by which alternative splicing is regulated. Alternative splicing is most commonly controlled by RNA binding proteins that bind to sequence elements called enhancers and silencers (Nilsen and Graveley 2010). Splicing regulators bound to these enhancers or silencers are thought to either recruit or inhibit assembly or activity of spliceosomal components at nearby splice sites. The best-characterized splicing regulator proteins are the SR and hnRNP protein families. SR proteins primarily bind to enhancer sequences in exons where they activate adjacent splice sites, while hnRNPs have mostly been shown to suppress splicing when bound to intronic silencers. In addition to SR and hnRNPs proteins, several other splicing regulators have been identified that function in a tissue specific manner (Chen and Manley 2009). The mammalian proteins NOVA1 and NOVA2 (collectively named here as NOVA) are perhaps the best-characterized splicing regulators to date. NOVA1/2 encode RNA binding proteins with three KH-domains that recognize clusters of YCAY repeats. Over the past decade, several hundred splicing events have been shown to be regulated by NOVA1/2 (Ule et al. 2005, 2006; Licatalosi et al. 2008). A comparison of the locations of the NOVA1/2 binding sites with NOVA-regulated splicing events has revealed a stereotypical “RNA map” for NOVA1/2. Specifically, regions upstream of exons where NOVA inhibits splicing and regions downstream from exons where NOVA activates splicing were enriched with NOVA binding sites (Ule et al. 2006; Licatalosi et al. 2008). Similar “RNA maps” that link the position of binding sites to typical activities of the regulatory proteins have also been developed for mammalian FOX1/2 (Zhang et al. 2008; Yeo et al. 2009), PTB (Xue et al. 2009), and four D. melanogaster hnRNP proteins (Blanchette et al. 2009). Such maps, splicing expression data, and RNA sequence motifs have recently been used to predict regulated tissue-specific splicing changes in mouse, strongly supporting the existence of a splicing code (Wang and Burge 2008; Barash et al. 2010; Zhang et al. 2010), a decipherable sequence-based information system that dictates the splicing pattern of a given pre-mRNA under a specific condition. Though considerable progress has been made, interpreting this code remains a formidable task in the field. In particular, it is unclear how the mouse splicing code can be applied to different species, especially distantly related organisms such as Drosophila. Moreover, the extent to which the RNA maps of individual splicing regulators are static or plastic throughout evolution has been unknown. We were interested in exploring the conservation of the splicing code between distantly related organisms. As a first step in this process, we sought to generate an RNA map of Pasilla (PS) (Seshaiah et al. 2001), the D. melanogaster ortholog of NOVA1/2. To identify PS-regulated exons, we used RNA-seq (Wold and Myers 2008) to identify splicing events that changed upon depletion of PS by RNAi. We conclude that the RNA map of PS and NOVA1/2 is highly conserved between mammals and insects.

Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila

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.

Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes

Abstract: Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs∗13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.

Using VAAST to Identify an X-Linked Disorder Resulting in Lethality in Male Infants Due to N-Terminal Acetyltransferase Deficiency

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.

High-resolution human cytomegalovirus transcriptome

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.

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain

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.

G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms

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

Association of TALS Developmental Disorder with Defect in Minor Splicing Component U4atac snRNA

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.

HSPC117 is the essential subunit of a human tRNA splicing ligase complex.

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.

Regulation of alternative splicing by the core spliceosomal machinery

Abstract: Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B' self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B' in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.

Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa

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.

A unique spectrum of somatic PIK3CA (p110α) mutations within primary endometrial carcinomas

Abstract: Purpose: The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and nonendometrioid endometrial cancer (NEEC). Experimental Design: We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wild type and mutant constructs in U2OS cells and measuring levels of AKTSer473 phosphorylation. Results: Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1–7 and half were in exons 9 and 20. The exons 1–7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93, and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKTSer473 compared to wild-type p110α. Overall, 62% of exons 1–7 PIK3CA mutants and 64% of exons 9–20 PIK3CA mutants were activating; 72% of exon 1–7 mutations have not previously been reported in endometrial cancer. Conclusions: Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway. Clin Cancer Res; 17(6); 1331–40. ©2011 AACR.

In vivo regulation of amyloid precursor protein neuronal splicing by microRNAs

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.

Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis

Abstract: Hajdu-Cheney syndrome is a rare autosomal dominant skeletal disorder with facial anomalies, osteoporosis and acro-osteolysis. We sequenced the exomes of six unrelated individuals with this syndrome and identified heterozygous nonsense and frameshift mutations in NOTCH2 in five of them. All mutations cluster to the last coding exon of the gene, suggesting that the mutant mRNA products escape nonsense-mediated decay and that the resulting truncated NOTCH2 proteins act in a gain-of-function manner.

KIF1A, an Axonal Transporter of Synaptic Vesicles, Is Mutated in Hereditary Sensory and Autonomic Neuropathy Type 2

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.

Critical association of ncRNA with introns

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.

A natural antisense transcript at the Huntington’s disease repeat locus regulates HTT expression

Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). Relatively little attention has been directed to the genomic features of the antisense strand at the HD locus, though the presence of a transcript from this strand has been suggested by a survey of the entire transcriptome and the existence of several EST tags. In this study, we identified huntingtin antisense (HTTAS), a natural antisense transcript at the HD repeat locus that contain the repeat tract. HTTAS is 5' capped, poly (A) tailed and contains three exons, alternatively spliced into HTTAS_v1 (exons 1 and 3) and HTTAS_v2 (exons 2 and 3). Exon 1 includes the repeat. HTTAS_v1 has a weak promoter, and is expressed at low levels in multiple tissue types and throughout the brain. Reporter assays indicate that while efficient promoter activity requires a short repeat, repeat expansion reduces promoter efficiency. Consistent with the reporter assays, levels of HTTAS_v1 are reduced in human HD frontal cortex. In cell systems, overexpression of HTTAS_v1 specifically reduces endogenous HTT transcript levels, while siRNA knockdown of HTTAS_v1 increases HTT transcript levels. Minigene constructs of the HD locus confirm the regulatory effect of HTTAS_v1 on HTT, and demonstrate that the effect is dependent on repeat length and is at least partially Dicer dependent. Together, these findings provide strong evidence for the existence of a gene antisense to HTT, with properties that include regulation of HTT expression.