Internal Exons Research Articles

Global Promotion of Alternative Internal Exon Usage by mRNA 3′ End Formation Factors

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

Origins and impacts of new mammalian exons.

Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species- and lineage-specific exons. Most new exons derived from unique rather than repetitiveintronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' UTRs and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues in which the exon was included. Increased expression correlated with the level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Altogether, these findings suggest that increased splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing altergene expression between tissues and between species.

Splicing of designer exons informs a biophysical model for exon definition.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3′ end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.

Detection of internal exon deletion with exon Del.

BackgroundExome sequencing allows researchers to study the human genome in unprecedented detail. Among the many types of variants detectable through exome sequencing, one of the most over looked types of mutation is internal deletion of exons. Internal exon deletions are the absence of consecutive exons in a gene. Such deletions have potentially significant biological meaning, and they are often too short to be considered copy number variation. Therefore, to the need for efficient detection of such deletions using exome sequencing data exists.ResultsWe present ExonDel, a tool specially designed to detect homozygous exon deletions efficiently. We tested ExonDel on exome sequencing data generated from 16 breast cancer cell lines and identified both novel and known IEDs. Subsequently, we verified our findings using RNAseq and PCR technologies. Further comparisons with multiple sequencing-based CNV tools showed that ExonDel is capable of detecting unique IEDs not found by other CNV tools.ConclusionsExonDel is an efficient way to screen for novel and known IEDs using exome sequencing data. ExonDel and its source code can be downloaded freely at https://github.com/slzhao/ExonDel.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-332) contains supplementary material, which is available to authorized users.

Comparing change-point location in independent series

We are interested in the comparison of the positions of the change-points in the segmentation of independent series. We consider a Bayesian framework with conjugate priors to perform exact inference on the change-point model. This work is motivated by the comparison of transcript boundaries in yeast grown under different conditions. When comparing two series, we derive the posterior credibility interval of the shift between the locations. When comparing more than two series, we compute the posterior probability for a given change-point to have the same location in all series. All calculations are made in an exact manner in a quadratic time. The performances of those approaches are assessed via a simulation study. When applied to yeast genes, this approach reveals different behavior between internal and external exon boundaries.

EXONSAMPLER: a computer program for genome-wide and candidate gene exon sampling for targeted next-generation sequencing.

The computer program EXONSAMPLER automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next-generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User-adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of EXONSAMPLER to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon-capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16,000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.

The BEL1-like family of transcription factors in potato

BEL1-type proteins are ubiquitous plant transcription factors in the three-amino-acid-loop-extension superfamily. They interact with KNOTTED1-like proteins, and function as heterodimers in both floral and vegetative development. Using the yeast two-hybrid system with POTATO HOMEOBOX1 (POTH1) as the bait, seven BEL1-type proteins were originally identified. One of these genes, designated StBEL5, has transcripts that move long distances in the plant and enhance tuberization and root growth. Using the potato genome database, 13 active BEL1-like genes were identified that contain the conserved homeobox domain and the BELL domain, both of which are essential for the function of BEL1-type proteins. Phylogenetic analysis of the StBEL family demonstrated a degree of orthology with the 13 BEL1-like genes of Arabidopsis. A profile of the gene structure of the family revealed conservation of the length and splicing patterns of internal exons that encode key functional domains. Yeast two-hybrid experiments with KNOTTED1-like proteins and the new StBELs confirmed the interactive network between these two families. Analyses of RNA abundance patterns clearly showed that three StBEL genes, BEL5, -11, and -29, make up approximately two-thirds of the total transcript values for the entire family. Among the 10 organs evaluated here, these three genes exhibited the 12 greatest transcript abundance values. Using a phloem-transport induction system and gel-shift assays, transcriptional cross-regulation within the StBEL family was confirmed. Making use of the potato genome and current experimental data, a comprehensive profile of the StBEL family is presented in this study.

The evolution of the coding exome of the Arabidopsis species--the influences of DNA methylation, relative exon position, and exon length.

BackgroundThe evolution of the coding exome is a major driving force of functional divergence both between species and between protein isoforms. Exons at different positions in the transcript or in different transcript isoforms may (1) mutate at different rates due to variations in DNA methylation level; and (2) serve distinct biological roles, and thus be differentially targeted by natural selection. Furthermore, intrinsic exonic features, such as exon length, may also affect the evolution of individual exons. Importantly, the evolutionary effects of these intrinsic/extrinsic features may differ significantly between animals and plants. Such inter-lineage differences, however, have not been systematically examined.ResultsHere we examine how DNA methylation at CpG dinucleotides (CpG methylation), in the context of intrinsic exonic features (exon length and relative exon position in the transcript), influences the evolution of coding exons of Arabidopsis thaliana. We observed fairly different evolutionary patterns in A. thaliana as compared with those reported for animals. Firstly, the mutagenic effect of CpG methylation is the strongest for internal exons and the weakest for first exons despite the stringent selective constraints on the former group. Secondly, the mutagenic effect of CpG methylation increases significantly with length in first exons but not in the other two exon groups. Thirdly, CpG methylation level is correlated with evolutionary rates (dS, dN, and the dN/dS ratio) with markedly different patterns among the three exon groups. The correlations are generally positive, negative, and mixed for first, last, and internal exons, respectively. Fourthly, exon length is a CpG methylation-independent indicator of evolutionary rates, particularly for dN and the dN/dS ratio in last and internal exons. Finally, the evolutionary patterns of coding exons with regard to CpG methylation differ significantly between Arabidopsis species and mammals.ConclusionsOur results suggest that intrinsic features, including relative exonic position in the transcript and exon length, play an important role in the evolution of A. thaliana coding exons. Furthermore, CpG methylation is correlated with exonic evolutionary rates differentially between A. thaliana and animals, and may have served different biological roles in the two lineages.

P.9.3 Repair of mutant nebulin transcripts by exon exchange

The nebulin gene (NEB) has 183 exons encoding transcripts up to 26 kb in length. Mutations found in NEB are dispersed throughout the gene. Mutations cause autosomal recessive nemaline myopathy, distal myopathy and core-rod myopathy, for which no therapy is available. The size of NEB limits the options of gene therapy development. Thus, our research has focused on methods correcting the mutation carrying transcripts. The exon exchange method can be developed to exchange a mutated exon by spliceosome mediated RNA trans-splicing occurring between the target pre-mRNA and a carefully designed therapy molecule, PTM (Pre-Trans-splicing Molecule), carrying the wild type exon. We have previously obtained successful results from our 3′ and internal exon exchange experiments using a minigene target in cell cultures. In our latest experiment we targeted the last exons of the nebulin transcript produced by C2C12 mouse myoblasts. This is the first time the endogenous nebulin has been used as a target for exon exchange. Our preliminary results indicate successful, but weak trans-splicing at the RNA level. By combining the knowledge gathered from the internal exon exchange experiments and the modification of the endogenous transcript, we are now planning a new experiment in which we target an internal exon of endogenous mutated nebulin transcripts in vivo using a zebrafish model for nemaline myopathy. The fish has a splice site mutation causing abnormal splicing of nebulin exon 43. Zebrafish nebulin has very small introns which make selecting PTM binding sites in the mutated area challenging. We have now designed five different PTM constructs targeted to the mutated region of zebrafish nebulin. The PTMs will first be tested in cell cultures targeting zebrafish nebulin minigene transcripts and the successful PTMs will be used in the in vivo experiments. This will be the first attempt to correct a mutated nebulin pre-mRNA in vivo.

Computational prediction and characterisation of ubiquitously expressed new splice variant of Prkaca gene in mouse

Prkaca gene of mouse encodes for a cAMP dependent protein kinase catalytic alpha subunit. PKA occurs naturally as a 4-membered structure having two regulatory (R) and two catalytic (C) subunits each encoded by separate gene. Alternatively spliced two transcript variants are known for the Prkaca gene, which encode for two isoforms of PKA C-subunits, namely Cα1 and Cα2. These isoforms arise as a result of alternative splicing of the first coding exon with the internal exons. We have identified a new transcript variant using combinatorial approach of bioinformatics and molecular biology techniques involving RT-PCR, semi-nested PCR and sequencing. The new transcript variant encoding Cα3 isoform has N-terminus that differs from Cα1 and Cα2 isoforms. Cα3 isoform also arise as a result of alternative splicing of first coding exon with the internal exon. Newly identified transcript is expressed ubiquitously in different tissues examined.

Pre-mRNA splicing is a determinant of nucleosome organization

Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5′ splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5′ splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization.

Pre-mRNA Splicing Is a Determinant of Nucleosome Organization

Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5′ splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5′ splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization.

Detection of an Unbalanced Ratio Between WT1 Isoforms in Acute Myeloid Leukemia and Its Correlation with Molecular Abnormalities.

AbstractAbstract 2500Wilms’ tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity.The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results:KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion:Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures:No relevant conflicts of interest to declare.

HEXEvent: a database of Human EXon splicing Events

HEXEvent (http://hexevent.mmg.uci.edu) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

Exclusion of Exon 2 Is a Common mRNA Splice Variant of Primate Telomerase Reverse Transcriptases

Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3′ half of the coding region. We were interested in examining splicing of the 5′ half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20–40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

Novel splicing events and post-transcriptional regulation of human estrogen receptor α E isoforms

Expression of the estrogen receptor α (ERα) gene is subject to complex regulation. To elucidate the mechanisms of this regulation, the genomic organization and the physiological role of the multiple 5′-untranslated regions (5′-UTRs) must be determined. Here, we investigated the expression and splicing patterns of the human ERα E isoforms. We identified two novel untranslated exons, N1 and N2, in the 5′-region of the human ERα gene and multiple E isoform mRNA variants generated by alternative usage of non-coding internal exons. Expression of the N1-containing variants was observed only in the human breast adenocarcinoma cell line, MCF7, while the N2-containing variants were expressed in the adult liver and MCF7 cells. We examined post-transcriptional regulation of the variant mRNAs using luciferase reporter assays and quantitative PCR. The insertion of untranslated internal exons into the 5′-UTRs of the E isoforms reduced their translation efficiency, but barely influenced mRNA turnover. Our results indicate that the genomic organization of the human ERα gene and the splicing profiles of the human ERα E isoforms are more complicated than previously reported. Furthermore, the 5′-UTRs of the E isoforms post-transcriptionally control human ERα expression mainly through translational repression.

Position-dependent correlations between DNA methylation and the evolutionary rates of mammalian coding exons

DNA cytosine methylation is a central epigenetic marker that is usually mutagenic and may increase the level of sequence divergence. However, methylated genes have been reported to evolve more slowly than unmethylated genes. Hence, there is a controversy on whether DNA methylation is correlated with increased or decreased protein evolutionary rates. We hypothesize that this controversy has resulted from the differential correlations between DNA methylation and the evolutionary rates of coding exons in different genic positions. To test this hypothesis, we compare human-mouse and human-macaque exonic evolutionary rates against experimentally determined single-base resolution DNA methylation data derived from multiple human cell types. We show that DNA methylation is significantly related to within-gene variations in evolutionary rates. First, DNA methylation level is more strongly correlated with C-to-T mutations at CpG dinucleotides in the first coding exons than in the internal and last exons, although it is positively correlated with the synonymous substitution rate in all exon positions. Second, for the first exons, DNA methylation level is negatively correlated with exonic expression level, but positively correlated with both nonsynonymous substitution rate and the sample specificity of DNA methylation level. For the internal and last exons, however, we observe the opposite correlations. Our results imply that DNA methylation level is differentially correlated with the biological (and evolutionary) features of coding exons in different genic positions. The first exons appear more prone to the mutagenic effects, whereas the other exons are more influenced by the regulatory effects of DNA methylation.

Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: “co-transcriptional splicing.” Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a “first transcribed, first spliced” rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.

T.P.25 Development of the exon exchange method for repair of mutant nebulin transcripts

Abstract The nebulin gene (NEB) has 183 exons encoding transcripts up to 26 kb in length. Mutations found in NEB are dispersed throughout the gene, i.e. no mutational hot spots are evident. Mutations cause autosomal recessive nemaline myopathy, distal nebulin myopathy and core-rod myopathy, for which no therapy is available. The size of NEB limits the options of gene therapy development. Thus, our research has focused on methods correcting the mutation carrying transcripts. Targeted exon exchange requires the spliceosome provided by the cell in addition to the target pre-mRNA and pre-trans-splicing molecules (PTMs) both produced from expression vectors transfected into C2C12 mouse myoblasts. We have developed wild type and mutant NEB minigenes for production of target pre-mRNA and PTMs for internal exon exchange of one or several NEB exons at a time. We have previously obtained successful results from our 3′ exon exchange and first internal exon exchange experiments and continue to develop the internal exon exchange technique further. NEB introns are usually short and binding of PTM can interfere with exon recognition. We have designed PTMs targeted to larger introns on both sides of an area of two exons close to each other to overcome this problem. Exon exchange is assessed by RT-PCR and sequencing and at the protein level by Western blotting and immunofluorescence staining. Our preliminary results from co-transfection of mutant minigenes and PTMs indicate successful, but weak trans-splicing at the RNA level. Immunofluorescence staining of co-transfected C2C12 cells show protein production in some cells, which may indicate successful trans-splicing, but the results need to be confirmed by western blotting. Being able to exchange several exons at a time from the 183-exon-containing NEB at the RNA level would be an advantage in therapy as the same therapy molecule would be useful for patients with mutations in any of the exons in the exchanged area.

Microarray-Based Capture of Novel Expressed Cell Type–Specific Transfrags (CoNECT) to Annotate Tissue-Specific Transcription inDrosophila melanogaster

Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated but also to identify potential novel, unannotated exons of genes, which may be additional targets of mutation in disease states or while performing mutagenic screens. We have developed a microarray enrichment methodology followed by long-read, next-generation sequencing for identification of unannotated transcript isoforms expressed in two Drosophila tissues, the ovary and the testis. Even with limited sequencing, these studies have identified a large number of novel transcription units, including 5′ exons and extensions, 3′ exons and extensions, internal exons and exon extensions, gene fusions, and both germline-specific splicing events and promoters. Additionally, comparing our capture dataset with tiling array and traditional RNA-seq analysis, we demonstrate that our enrichment strategy is able to capture low-abundance transcripts that cannot readily be identified by the other strategies. Finally, we show that our methodology can help identify transcriptional signatures of minority cell types within the ovary that would otherwise be difficult to reveal without the CoNECT enrichment strategy. These studies introduce an efficient methodology for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation.