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

ABSTRACT: The Drosophila melanogaster gene Dscam can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, it remained unexplored how many isoforms are indeed expressed, whether the splicing choice between the clusters is independent and why the diversity encoded in the gene locus seems to be beyond necessity. To address these questions, we developed IsomSeq, a novel multiple segment sequencing method that allows to directly quantify the alternatively spliced exon combination of Dscam isoforms within an entire cellular and organismal transcriptome.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE45167 | GEO | 2013/06/27

SECONDARY ACCESSION(S): PRJNA193150

REPOSITORIES: GEO

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

Project description:The Drosophila melanogaster gene Dscam can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, it remained unexplored how many isoforms are indeed expressed, whether the splicing choice between the clusters is independent and why the diversity encoded in the gene locus seems to be beyond necessity. To address these questions, we developed IsomSeq, a novel multiple segment sequencing method that allows to directly quantify the alternatively spliced exon combination of Dscam isoforms within an entire cellular and organismal transcriptome. We sequenced the dscam repertoire of adult brain, S2 cell line, and 5 different developmental stages (embryo, L1, L2, L3, pupa)

2013-06-27 | E-GEOD-45167 | biostudies-arrayexpress

Memory consolidation in honey bees is enhanced by down-regulation of Down Syndrome Cell Adhesion Molecule and changes its alternative splicing

Project description:Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of invertebrate Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in differentiated brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation possibly to alter neuronal connections for memory retention.

2024-01-05 | GSE244051 | GEO

DSCAM-AS1-driven proliferation of breast cancer cells involves regulation of alternative exon splicing and 3’-end usage

Project description:Background: DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B and HER2-positive Breast Cancer (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). Method: To decipher its function, DSCAM-AS1 expression was measured by qRT-PCR in tissue samples from 93 BC patients in addition to a meta-analysis of 30 gene expression datasets, together with the evaluation of its association with clinical data. By computational analyses of our RNA-Seq in MCF-7 cells, we investigated the DSCAM-AS1 knock-down effects at both gene and isoform levels Results: We confirmed DSCAM-AS1 overexpression in high grade Luminal A, B and HER2+ BCs and found a significant correlation with disease relapse. 908 genes were regulated by DSCAM-AS1-silencing, primarily involved in cell cycle and inflammatory response. Noteworthy, the analysis of alternative splicing and isoform regulation revealed 2,085 splicing events regulated by DSCAM-AS1, enriched in differential polyadenylation sites and 3’UTR shortening events. Finally, the DSCAM-AS1-interacting splicing factor hnRNPL was predicted as the most enriched RBP for exon skipping and 3’UTR events. Conclusion: The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer.

2020-05-15 | GSE150591 | GEO

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

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

| S-EPMC3715862 | biostudies-literature

Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation

Project description:DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we further found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the beta-importin family, via a conserved nuclear localization signal. The DSCAM ICD is released by gamma-secretase dependent cleavage and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA-sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation, synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21 our findings may help to uncover novel mechanisms contributing to intellectual disability in Down syndrome.

2019-02-08 | GSE122568 | GEO

Sphingolipid-dependent Dscam sorting regulates axon segregation

Project description:Neurons are highly polarized cells with distinct protein compositions in axonal and dendritic compartments. Cellular mechanisms controlling polarized protein sorting have been described for mature nervous system but little is known about the segregation in newly differentiated neurons. In a forward genetic screen for regulators of Drosophila brain circuit development, we identified mutations in <span style="color: rgb(54, 54, 54); font-style: normal; font-weight: 400; background-color: rgb(245, 245, 245);">Serine Palmitoyltransferase </span>(SPT), an evolutionary conserved enzyme in sphingolipid biosynthesis. Here we show that reduced levels of sphingolipids in SPT mutants cause axonal morphology defects similar to loss of cell recognition molecule Dscam. Loss- and gain-of-function studies show that neuronal sphingolipids are critical to prevent aggregation of axonal and dendritic Dscam isoforms, thereby ensuring precise Dscam localization to support axon branch segregation. Furthermore, SPT mutations causing neurodegenerative HSAN-I disorder in humans also result in formation of stable Dscam aggregates and axonal branch phenotypes in Drosophila neurons, indicating a causal link between developmental protein sorting defects and neuronal dysfunction.

2019-03-29 | MTBLS825 | MetaboLights

Transposable element exonization by non-canonical splicing generates a diversity reservoir of functional protein isoforms

Project description:mRNA splicing enlarges the diversity of the protein repertoire, mainly through alternative exon retention or skipping. Although non-canonical splicing variants that exonize intronic regions were recently identified at the mRNA level, their contribution to the cellular proteome remains unclear. Here, we describe a population of 1300 unannotated protein isoforms generated by non-canonical splicing between exons and transposable elements (TE) using a combination of transcriptome assembly, ribosome profiling, and mass spectrometry. Despite being shorter and expressed at lower levels, their translation efficiency is similar to that of canonical isoforms, and they are shared between individuals. Functional analyses of 5 different non-canonical isoforms show stable expression, specific intracellular localization, and modified functions, as compared to the corresponding canonical isoforms. Non-canonical isoforms derive mainly from evolutionarily ancient genes and are a preferential source of alternative splicing upon which natural selection can act. We conclude that recently exonized TE isoforms are potentially functional and represent a diversity reservoir of novel protein isoforms.

2024-12-12 | GSE234223 | GEO

DSCAM-AS1-driven proliferation of breast cancer cells involves regulation of alternative exon splicing and 3’-end usage

Project description:DSCAM-AS1-driven proliferation of breast cancer cells involves regulation of alternative exon splicing and 3’-end usage

| PRJNA632880 | ENA

RNA-seq analysis dataset: Cerebellum tissues from wild type and Dscam del17/del17 mice at P21

Project description:The cytoplasmic domain of DSCAM has been reported to exhibit transcriptional activity on genes involved in neuronal wiring in HEK293 cells. Thus, we addressed the possibility that deregulation of the CF synapse in Dscamdel17/del17 mice is caused by the reduced expression of CF synaptogenesis-related genes, by using RNA-seq analyses using the P21 whole cerebellum of WT and Dscamdel17/del17 mice. Expression levels of most of the genes, except for Nefl, Mal, Fat2, and Fat1, were not significantly different between the WT and Dscamdel17/del17 mice, probably because depletion of Dscam in the cerebellum affects only a few cell types, including Purkinje cells. We also confirmed the unchanged expression of Purkinje cell-specific genes (and their encoded proteins), such as Cacna1a (Cav2.1), Sema7a and Sema3a, Grm1 (mGluR1) and Prkcg (protein kinase Cγ [PKCγ]), Grid2 (GluD2), and Adgrb3 (Bai3) between the WT and Dscamdel17/del17 mice. These results raise the possibility that DSCAM in Purkinje cells regulates synapse formation and function, independently of the regulation of gene expression of known CF synaptogenesis-related genes.

2023-11-30 | GSE190010 | GEO

The dynamic landscape of fission yeast meiosis alternative-splice isoforms

Project description:Alternative splicing increases the diversity of transcriptomes and proteomes in metazoans. The extent to which alternative splicing is active and functional in unicellular organisms is less understood. Here we exploit a single-molecule long-read sequencing technique and develop a computational tool, SpliceHunter, to characterize the transcriptome in the meiosis of fission yeast. We reveal 17017 alternative splicing events in 19741 novel isoforms at different stages of meiosis, including antisense and read-through transcripts. Intron retention is the major type of alternative splicing, followed by “alternate intron in exon”. 887 novel transcription units are detected; 60 of the predicted proteins show homology in other species and form theoretical stable structures. We compare the dynamics of novel isoforms based on the number of supporting full-length reads with those of annotated isoforms and explore the translational capacity and quality of novel isoforms. The evaluation of these factors indicates that the majority of novel isoforms are unlikely to be both condition-specific and translatable but the possibility of functional novel isoforms is not excluded. Together, this study highlights the diversity and dynamics at the isoform level in the sexual development of fission yeast.

2016-11-10 | GSE79802 | GEO

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