New strategies for the identification of intronic variants related to splicing events in pancreas cancer [RNA-Seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE228844 | GEO | 2024/05/08
REPOSITORIES: GEO
Similar Datasets
Project description:Most clinical diagnostic settings and genomic research focus almost exclusively on coding regions and essential splice sites, mostly ignoring non-coding variants. Indeed, the investigation of intronic mis-splicing variants interpreting mechanisms to disease-associated splicing events requires both genomic and transcriptomic data. Unfortunately, there are not many datasets where both are available, leading to the understanding of intronic variants in diseases full of gaps. In this study, we present for the first time a full-length single nuclei RNA-sequencing (snRNA-Seq) approach improving the proper investigation of pathogenic mis-splicing intronic variants in pancreatic cancer showing its contribution to abnormal splicing changes and their transcriptional effects. Finally, we discuss the demands of further machine learning-based methods to process the growing number of single cell data and to enhance the precision and recall.
2024-05-10 | GSE229006 | GEO
Project description:Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA, remain elusive. Results: Employing in silico analysis we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3’-splice site associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.
2020-10-28 | PXD022204 | Pride
Project description:Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure and evolutionary conservation features. RegSNPs-intron shows excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of regSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.
2019-09-30 | GSE138130 | GEO
Project description:BACKGROUND: Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. RESULTS: Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5-5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. CONCLUSIONS: Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program. Keywords: Time course study – effect of androgen on gene expression
2007-01-01 | GSE5345 | GEO
Project description:Approximately half of all microRNAs reside within intronic regions and are often co-transcribed with their host genes. However, most studies on intronic microRNAs focus on individual microRNAs, and conversely most studies on protein-coding and non-coding genes frequently ignore any intron-derived microRNAs. We hypothesize that the individual components of such multi-genic loci may play cooperative or competing roles in driving disease progression, and that examining the combinatorial effect of these components would uncover deeper insights into their functional importance. To address this, we perform systematic analyses of intronic microRNA:host loci in colon cancer. We observe that the FTX locus, comprising of a long non-coding RNA FTX and multiple intronic microRNAs, is highly upregulated in cancer and demonstrate that cooperativity within this multi-component locus promotes cancer growth. In addition, we show that FTX interacts with DHX9 and DICER and delineate its novel roles in regulating A-to-I RNA editing and microRNA expression. These results show for the first time that a long non-coding RNA can regulate A-to-I RNA editing, further expanding the functional repertoire of long non-coding RNAs. We further demonstrate the inhibitory effects of intronic miR-374b and -545 on the tumor suppressors PTEN and RIG-I to enhance the proto-oncogenic PI3K-AKT signaling. Finally, we show that intronic miR-421 may exert an autoregulatory effect on miR-374b and -545. Taken together, our data unveil the intricate interplay between intronic microRNAs and their host transcripts in the modulation of key signaling pathways and disease progression, adding new perspectives to the functional landscape of multi-genic loci.
2021-01-05 | PXD021823 | Pride
Project description:Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease- causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.
2023-05-19 | GSE185943 | GEO
Project description:To compare nuclear and cytoplasmic RNA from a single cell type, free of cross-contamination, we studied the oocyte of the frog Xenopus tropicalis, a giant cell with an equally giant nucleus. We isolated RNA from manually dissected nuclei and cytoplasm of mature oocytes and subjected it to deep sequencing. Cytoplasmic mRNA 10 consisted primarily of spliced exons derived from ~6700 annotated genes. Nearly all of these genes were represented in the nucleus by intronic sequences. However, unspliced nascent transcripts were not detected. Inhibition of transcription or splicing for 1M-bM-^@M-^S2 d had little or no effect on the abundance of nuclear intronic sequences, demonstrating that they are unusually stable. RTM-bM-^@M-^SPCR analysis showed that these stable intronic sequences are transcribed from the coding strand and that a given intron can be processed into more than one 15 molecule. Stable intronic sequence RNA (sisRNA) from the oocyte nucleus constitutes a new class of noncoding RNA. sisRNA is detectable by RTM-bM-^@M-^SPCR in samples of total RNA from embryos up to the mid-blastula stage, when zygotic transcription begins. Storage of sisRNA in the oocyte nucleus and its transmission to the developing embryo suggest that it may play important regulatory roles during oogenesis and/or early embryogenesis. The individual germinal vesicle (GV) samples' log2 signals were compared pairwise with those from enucleated oocytes.
2012-11-26 | E-GEOD-41530 | biostudies-arrayexpress
Project description:Rare intronic variants of TCF7L2 arising by selective sweeps in an indigenous population from Mexico
| PRJEB13132 | ENA
Project description:Mutations that attenuate DNA repair by homologous recombination (HR) promote tumorigenesis and sensitize cells to chemotherapeutic agents that cause replication fork collapse, a phenotype known as “BRCAness.” BRCAness tumors arise from loss-of-function mutations in 22 genes. Of these genes, all but one (Cdk12) directly function in the HR repair pathway. Cdk12 phosphorylates Serine 2 of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeat, a modification that regulates transcription elongation, splicing, and cleavage/polyadenylation. Genome-wide expression studies suggest that Cdk12 depletion abrogates the expression of several HR genes relatively specifically, blunting HR repair. This observation suggests that Cdk12 mutational status may predict tumor sensitivity to targeted treatments against BRCAness, such as Parp 1 inhibitors, and that small-molecule inhibitors of Cdk12 may induce sensitization of otherwise HR-competent tumors to these treatments. Despite this growing clinical interest, the mechanism behind the apparent specificity of Cdk12 in regulating HR genes remains unknown. Here we find that Cdk12 globally suppresses intronic polyadenylation events, enabling the production of full-length gene products. Many HR genes harbor significantly more intronic polyadenylation sites compared to all expressed genes, and the cumulative effect of these sites accounts for the increased sensitivity of HR gene expression to Cdk12 loss. Finally, we find evidence that Cdk12 loss-of-function mutations cause increased intronic polyadenylation within HR genes in human tumors, suggesting that this mechanism is conserved. This work clarifies the biological function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.
2018-11-28 | GSE116017 | GEO
Project description:Purpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6M-NM-^T), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6M-NM-^T, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced. Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD). We report RNAseq data for two SM immunoprecipitation experiments and RNAseq datasets for the parental strain (BY4741), the prp40-1 mutant, and the rrp6M-NM-^T strain.
2013-10-11 | E-GEOD-49966 | biostudies-arrayexpress