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Unprecedented alternative splicing and 3 Mb of novel transcribed sequence leads to significant transcript diversity in the transcriptome of the human retina

ABSTRACT: The mRNA repertoire is specific to a given tissue type. This specificity, whether it is in the levels of gene expression or the splicing pattern of the transcripts, is important to study to increase our general knowledge of transcription and alternative splicing, as well as to identify novel coding sequence that can be important for elucidating the mechanisms of disease. We have performed RNA-Seq on three normal human retinal samples and have found a profound level of novel alternative splicing including novel exons, exon skipping, and 3’ and 5’ alternate splice sites. Additionally, we have identified hundreds of novel genes. With so many novel splicing events identified in the retinal transcriptome, we set out to determine if these novel features were indeed real. We developed a high throughput capture set to target 14,696 of the novel features. We found that 99% of the novel features do validate, including those initially detected at a depth of coverage as low as one. These results confirm that the transcripts expressed in the retina are much more diverse than previously identified. The complete human retinal transcriptome described here will be useful for investigators studying multiple aspects of retinal biology and disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40524 | GEO | 2013/07/30

SECONDARY ACCESSION(S): PRJNA174224

REPOSITORIES: GEO

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Unprecedented alternative splicing and 3 Mb of novel transcribed sequence leads to significant transcript diversity in the transcriptome of the human retina

Project description:The mRNA repertoire is specific to a given tissue type. This specificity, whether it is in the levels of gene expression or the splicing pattern of the transcripts, is important to study to increase our general knowledge of transcription and alternative splicing, as well as to identify novel coding sequence that can be important for elucidating the mechanisms of disease. We have performed RNA-Seq on three normal human retinal samples and have found a profound level of novel alternative splicing including novel exons, exon skipping, and 3M-bM-^@M-^Y and 5M-bM-^@M-^Y alternate splice sites. Additionally, we have identified hundreds of novel genes. With so many novel splicing events identified in the retinal transcriptome, we set out to determine if these novel features were indeed real. We developed a high throughput capture set to target 14,696 of the novel features. We found that 99% of the novel features do validate, including those initially detected at a depth of coverage as low as one. These results confirm that the transcripts expressed in the retina are much more diverse than previously identified. The complete human retinal transcriptome described here will be useful for investigators studying multiple aspects of retinal biology and disease. Characterization of alternative splicing in the normal human retinal transcriptome.

2013-07-30 | E-GEOD-40524 | biostudies-arrayexpress

Next-generation sequencing identifies mRNAs targets that experience significant changes in alternative splicing upon PQBP1 knockdown in mouse embryonic cortical neurons

Project description:PQBP1 is a highly conserved protein closely related to neurodegenerative disorders. We identified PQBP1 as an important alternative splicing effector necessary for maintaining normal neuron functions in the brain. In order to explore PQBP1's functions in alternative splicing regulation and neuronal activities, we systematically profiled the alternative splicing targets of PQBP1 in mouse embryonic cortical neurons by RNA-seq. The mRNAs whose alternative splicing are affected by PQBP1 showed tissue-specific functional enrichment especially in neurite outgrowth, with strong Gene Ontology (GO) enrichments for neuron projection development/morphogenesis, dendrite development and axonogenesis. PQBP1's alternative splicing targets are also functionally enriched in RNA splicing, chromatin modification, and ARF signal transduction. We applied RNA-seq to compare the transcriptomes of mock and PQBP1 knockdown mouse embryonic cortical neuron samples.

2013-03-15 | E-GEOD-44402 | biostudies-arrayexpress

Next-generation sequencing identifies mRNAs targets that experience significant changes in alternative splicing upon PQBP1 knockdown in mouse embryonic cortical neurons

2013-03-15 | GSE44402 | GEO

Comparative Analysis of RNA-Seq Alignment Algorithms and the RNA-Seq Unified Mapper (RUM).

Project description:A critical task in high throughput sequencing is aligning millions of short reads to a reference genome. Alignment is especially complicated for RNA sequencing (RNA-Seq) because of RNA splicing. A number of RNA-Seq algorithms are available, and claim to align reads with high accuracy and efficiency while detecting splice junctions. RNA-Seq data is discrete in nature; therefore with reasonable gene models and comparative metrics RNA-Seq data can be simulated to sufficient accuracy to enable meaningful benchmarking of alignment algorithms. The exercise to rigorously compare all viable published RNA-Seq algorithms has not previously been performed. RESULTS: We developed an RNA-Seq simulator that models the main impediments to RNA alignment, including alternative splicing, insertions, deletions, substitutions, sequencing errors, and intron signal. We used this simulator to measure the accuracy and robustness of available algorithms at the base and junction levels. Additionally, we used RT-PCR and Sanger sequencing to validate the ability of the algorithms to detect novel transcript features such as novel exons and alternative splicing in RNA-Seq data from mouse retina. A pipeline based on BLAT was developed to explore the performance of established tools for this problem, and to compare it to the recently developed methods. This pipeline, the RNA-Seq Unified Mapper (RUM) performs comparably to the best current aligners and provides an advantageous combination of accuracy, speed and usability. RNA-Seq of mouse retinal RNA, as described.

2011-08-03 | E-GEOD-26248 | biostudies-arrayexpress

Splicing factor PRPF31 retinitis pigmentosa (RP11) is caused by disrupted alternative splicing programmes for genes implicated in pre-mRNA splicing, cellular adhesion and ciliogenesis

Project description:Mutations in pre-mRNA processing factors (PRPFs) cause autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause retinal disease. We have generated transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical-basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene-editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

2018-08-20 | PXD010821 | Pride

Quantitative microarray profiling of alternative splicing in mouse tissues

Project description:We describe the application of a new microarray platform, which combines information from exon body and splice-junction probes, to analyze the regulation of 3126 alternative splicing events in ten mouse tissues. The details of the methods and algorithms are described in this paper: Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform (Molecular Cell, Dec., 2004) Keywords = A new quantitative alternative splicing microarray platform

2010-05-27 | E-GEOD-2054 | biostudies-arrayexpress

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

Project description:The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926A>C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

2024-03-01 | PXD043645 | Pride

Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes.

Project description: Not available

| S-EPMC3924432 | biostudies-literature

Analysis of the implication of the DBIRD complex (DBC1 and ZNF326/ZIRD) in gene expression and alternative splicing

Project description:Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription and the rate of transcript elongation has a profound effect on splicing. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle that represents its functional form, and the composition of which determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles and thereby identified Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD. These proteins are subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. These data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with regulation of alternative splicing. HEK 293 cells were transfected with CTR-, DBC1- and ZIRD (ZNF326)- specific stealth siRNAs. Total RNAs from three independent experiments were extracted after 48 hours. All conditions were analyzed in triplicate.

2012-03-20 | E-GEOD-35480 | biostudies-arrayexpress

A Functional Network of Gastric-Cancer-Associated Splicing Events Controlled by Dysregulated Splicing Factors

Project description:Except for a few genes known to have oncogenic spliceforms in gastric cancer, the full scope of aberrant splicing in gastric oncogenesis remains unclear. In this study, we elucidated the alternative splicing events associated with gastric carcinogenesis and identified upstream regulators governing these events. We performed RNA-seq on 19 matched tumor/normal pairs and a panel of gastric cancer cell lines, and identified 361 tumor associated (TA) alternative splicing events (ASEs), which included known oncogenic ASEs in genes such as INSR, FGFR2, CD44 and KRAS. We further identified 8 splicing factors dysregulated in gastric tumors, the expression of which are correlated to the splice ratio of the TA ASEs. We thereby constructed a dysregulated splicing network in gastric cancer, consisting of alternative splicing changes correlated to their potential regulators. This network featured three potential regulatory modules centered around the splicing factors ESRP2, MBNL1 and PTPB1. Knockdown of each splicing factor led to the expected changes in splicing of approximately half of the ASEs assayed. Thus, dysregulation of the splicing factors indeed was the cause of many of the TA ASEs. Pathway enrichment analysis showed that genes affected by tumor associated splicing were pre-dominantly involved in cytoskeletal organization. Concordant with this, knockdown of the splicing factors regulating the TA ASEs led to the expected changes in trans-well migration activity. Thus, we have established for the first time a comprehensive splicing network that likely drives gastric tumor progression, based on which novel therapeutic targets can be identified for further evaluation.

| EGAS00001002256 | EGA

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