Alternative splicing isoforms in patient-derived hepatocellular carcinoma cells
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ABSTRACT:
PROVIDER: EGAS00001002697 | EGA |
REPOSITORIES: EGA
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Project description:The Plasmodium falciparum human malaria parasite genome is incompletely annotated and does not accurately represent the transcriptomic diversity of this species. To address this need, we performed long-read transcriptomic sequencing. To increase representation of mRNA isoforms expressed during intraerythrocytic stages, RNA was extracted from samples taken at time-points from synchronized cultures. 5 capped mRNA was enriched and converted to cDNA library. The cDNA libraries were sequenced on Nanopore and PacBio long-read platforms. Candidate non-redundant isoforms were annotated from each library dataset for creating a new transcript annotation catalog. Isoforms with alternative 5 ends represented the largest class of novel isoforms, although the majority of these do not seem to represent the products of alternative transcription initiation events. We suggest that most isoforms do not encode protein isoforms and instead represent RNA decay products.
2022-10-26 | GSE186109 | GEO
Project description:Genome-wide association studies (GWASs) have revealed thousands of associations in many complex traits and diseases. Previous studies suggest that a subset of associations are due to alterations in splicing; however, interpreting the effects of splicing on protein isoforms is hindered by limitations in defining full-length transcript isoforms using short-read RNA-seq data. Long-read RNA-seq represents a powerful approach to define and quantify transcript isoforms. In this study, we developed a novel approach that integrates information from GWAS, splicing QTL (sQTL), and PacBio long-read RNA-seq in a disease relevant model to infer the effects of sQTL on the ultimate protein isoform products they encode. Such information enables identification of genes potentially responsible for GWAS associations. As a proof-of-concept, we generated deep coverage (N=~22 million full-length reads) PacBio long-read RNAseq data on human fetal osteoblasts (hFOBs), a cell-line of relevance to the regulation of bone mineral density (BMD). We identified 68,326 protein-coding isoforms, including 17,375 (25%) which were novel. Next, we used Bayesian colocalization to identify 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes which colocalized with BMD associations (H4PP > 0.75). A total of 836 junctions with colocalizing sQTLs in 459 (of the 732) genes were expressed in hFOB long-read RNA-seq data. With these data, we formulated hypotheses regarding the potential mechanism of action of each sQTL. For example, we identified 7 junctions with colocalizing sQTLs (maximum H4PP = 0.98-0.99) in TPM2 for splice junctions between two nearly mutually exclusive exons, and two different transcript termination sites, making it impossible to interpret without long-read RNA-seq data. siRNA mediated knockdown in hFOBs showed two TPM2 isoforms with opposing effects on mineralization. Our results suggest that splicing is a major mechanism underlying GWAS associations and long-read proteogenomics data is critical to precisely define the protein isoforms that are produced from splicing alterations.
2023-03-01 | GSE224588 | GEO
Project description:Human preimplantation development is a complex process involving extensive remodeling of gene expression. However, the preimplantation embryo transcriptome has only been annotated using short-read sequencing, which fails to capture full-length mRNAs and associated isoform diversity. We present a novel human embryo transcriptome using integrated long- and short-read RNA sequencing data. Our analysis reveals a total of 110,212 novel isoforms transcribed from known genes containing either a novel combination of known splice sites or at least one novel splice site, and 17,964 isoforms transcribed from completely novel genes located either in antisense direction of known genes or in intergenic space.
2023-09-26 | GSE190547 | GEO
Project description:Human preimplantation development is a complex process involving extensive remodeling of gene expression. However, the preimplantation embryo transcriptome has only been annotated using short-read sequencing, which fails to capture full-length mRNAs and associated isoform diversity. We present a novel human embryo transcriptome using integrated long- and short-read RNA sequencing data. Our analysis reveals a total of 110,212 novel isoforms transcribed from known genes containing either a novel combination of known splice sites or at least one novel splice site, and 17,964 isoforms transcribed from completely novel genes located either in antisense direction of known genes or in intergenic space.
2023-09-26 | GSE190544 | GEO
Project description:Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. Keywords: Comparative genomic hybridiation within a 3 generation pedigree We used 14 individuals from the 3 generation CEPH/UTAH 1444 pedigree for our analysis of looking at heritability of differentially spliced exons within the family. 3 biological growths of individuals NA12739, NA12740, NA12750, and NA12751 were used and a single biological growth for the remaining 10 individuals.
2007-08-07 | E-GEOD-7952 | biostudies-arrayexpress
Project description:Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. Keywords: Comparative genomic hybridiation within a 3 generation pedigree
2007-08-08 | GSE7952 | GEO
Project description:Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms.In this work, we compared RNC-seq and Ribo-seq in the context of proteome identification, especially when identifying protein isoforms from AS. We also demonstrated that the single-molecule long read sequencing technique identified thousands of new splice variants and guided the MS identifications of new protein isoforms.
2022-06-22 | GSE198624 | GEO
Project description:Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. Here, we demonstrate a direct role for histone modifications in alternative splicing. We find distinctive histone modification signatures which correlate with splicing outcome in a set of human genes. Modulation of histone modifications causes splice site switching. The mechanism for histone-mediated splice site selection involves a histone mark which is read by a chromatin protein, which in turn recruits a splicing regulator. These results outline an adaptor system for reading of histone marks by the pre-mRNA splicing machinery.
2010-02-10 | GSE19373 | GEO
Project description:Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. Here, we demonstrate a direct role for histone modifications in alternative splicing. We find distinctive histone modification signatures which correlate with splicing outcome in a set of human genes. Modulation of histone modifications causes splice site switching. The mechanism for histone-mediated splice site selection involves a histone mark which is read by a chromatin protein, which in turn recruits a splicing regulator. These results outline an adaptor system for reading of histone marks by the pre-mRNA splicing machinery. To obtain an estimate of how many PTB-dependent alternative splicing events are regulated by SET2/MRG15-mediated recruitment of PTB, we carried out a genomewide comparative analysis of alternative splicing in hMSC cells depleted of either SETD2, MRG15 or PTB using specific siRNAs, or mock-depleted using a control siRNA.
2010-02-10 | E-GEOD-19373 | biostudies-arrayexpress
Project description:Alternative splicing is an essential mechanism for diversifying proteins, with different mature RNA isoforms producing different proteins with potentially distinct functions. Two major challenges in characterizing the cellular function of isoforms are the lack of both experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design. To address these gaps, we developed and methodically tested a strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs (gRNAs) that overlap exon-exon junctions in the mature RNA. We perform a high-throughput essentiality screen, numerous qPCR assays, and PacBio long read sequencing to affirm that our strategy works to specifically target the unique junctions of isoforms to robustly knockdown their RNA. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions in current GENCODE annotations and our predictions of gRNA efficacy, we estimate that this strategy can target 94% of all human isoforms, including 29,238 protein-coding and 9,638 lncRNA isoforms that are specifically targetable with a gRNA spanning a unique splice junction.
2023-09-13 | GSE242106 | GEO