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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

A proteogenomic atlas of the human neural retina

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2024-10-17 | PXD045187 | Pride

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.

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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.

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The BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1-mutated chronic lymphocytic leukemia

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| EGAS00001006771 | EGA

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

Developmental Transcriptome Dynamics of the Murine Retina

Project description:This study provides a comprehensive transcript analysis of the developing mouse retina with a focus on normalization, differentially expression, cell-type specific gene expression, transcription factor co-expression, alternative splicing, and novel transcript discovery. RNA-seq profiling was performed on four embryonic and eight postnatal time points during mouse retinal development and maturation. Alignment, transcript quantitation, normalization and differential expression, alternative splice usage, and novel transcript discovery was performed. Quantitative analysis revealed that 25,901 total transcripts encoding for 13,714 genes were expressed in the mouse retina between embryonic day 11 and postnatal day 28. Of these expressed transcripts, 12,075 were significantly differentially expressed (10,069 genes) at some point during development corresponding to ~73% of the expressed genes.

2017-12-07 | GSE101986 | GEO

An uncharacterized gene, C7orf26, identified from an autosomal dominant ocular disease functions in mRNA splicing

Project description:An uncharacterized gene, C7orf26 was expected to function with the integrator complex in mRNA splicing. To address the involvement of mRNA splicing, we performed microarray analysis of HeLa/C7orf26 siRNA cells and HeLa/non-specific (NS) and identified 700 transcripts that were downregulated by 50% or more in C7orf26 siRNA-treated HeLa cells. Gene set enrichment analysis showed that genes tagged with ‘alternative splicing’ or ‘splice variant’ occupy a large fraction of the downregulated genes.

2023-03-08 | GSE186323 | 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

Comprehensive characterization of transcript isoform in mouse retina with ONT long read single-cell RNA sequencing

Project description:The retina, a complex neural tissue, encompasses a vast diversity of over 100 distinct cell types, each characterized by unique features such as morphology, function, location, and transcriptomic profiles. However, the extent of mRNA alternative splicing within individual cell types in the retina remains largely uncharted territory. To bridge this knowledge gap, we employed single-cell RNA sequencing, utilizing both short-read and long-read high-throughput technologies. Our comprehensive analysis profiled the transcriptomes of 29,191 mouse retina cells, resulting in a dataset comprising 1.54 billion Illumina short reads and 1.40 billion Oxford Nanopore long reads. Remarkably, this exploration unveiled a staggering 44,325 transcript isoforms, with 38% of them representing entirely novel discoveries and 17% exhibiting cell-class-specific expression patterns. Intriguingly, while many isoforms were expressed across various cell types, their distribution often displayed variability among them. This research substantially enriches the catalog of transcript isoforms, laying the groundwork for further investigations into the role of alternative splicing in retinal biology and its implications for related diseases. It represents a crucial step toward unraveling the intricacies of retinal function and offers promising insights into the molecular mechanisms underlying retinal disorders.

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