HDBR1 OVEREXPRESSED CALU6 AND 293T CELLS VS. CONTROL CELLS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiScanSQ
ORGANISM(S): Homo sapiens
SUBMITTER: BING HAN
PROVIDER: E-MTAB-5347 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2019-04-17 | GSE117416 | GEO
Project description:Introns in pre-mRNAs must be spliced out prior to their translation. During splicing, introns are removed in the form of a lariat, in which the 5' end is linked to the 2' hydroxyl of an internal adenosine. Lariat degradation is initiated by an 2'-5' phosphodiester-specific RNA endonuclease which debranches these lariat RNAs to linear form. Deletion of the debranching enzyme is yeast results in the accumulation of lariat introns. We used this accumulation to identify spliced lariat introns on a genome-wide scale in S. cerevisiae using tiling microarrays. Keywords: two sample comparison, 3 biological replicates
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Project description:Transciptional profiling of Calu6 tumors comparing 4 pooled untreated tumors to 5-azacytodine or 5-azacytodine + MS-275 (combined therapy) treated tumors Three-condition experiment, untreated vs. 5-Azacytodine tumors, and untreated vs. 5-azacytodine + MS-275 tumors: 4 pooled untreated tumors; 4 5-Azacytodine treated, 4 5-Azacytodine + MS-275 treated. Study used a loop design with dye-swap.
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Project description:Gene splicing requires three basal genetic elements; the 3M-bM-^@M-^Y and 5M-bM-^@M-^Y splice sites and the branchpoint to which the 5M-bM-^@M-^Y intron termini is ligated to form a closed lariat during the splicing reaction. The 5M-bM-^@M-^Y and 3M-bM-^@M-^Y splice sites that define exon boundaries have been widely identified, revealing pervasive transcription and splicing of human genes. However, the locations of the third requisite element, the branchpoint, are still largely unknown. Here we employ two complementary approaches, targeted RNA sequencing and exoribonuclease digestion, to distil sequenced reads that traverse the lariat junction and, via non-conventional alignment, locate human branchpoint nucleotides. Alignments identify 88,748 branchpoints that correspond to 20% of known introns, with 76% supported by diagnostic sequence mismatch errors. This affords a first genome-wide analysis of branchpoints, describing their distribution, selection, and the existence of a diverse array of overlapping sequence motifs with distinct usage, evolutionary histories, and co-variation with distal splicing elements. The overlap of branchpoints with noncoding human genetic variation also indicates a notable contribution to disease. This annotation and analysis incorporates branchpoints into transcriptomic research and reflects a core role for this element in the regulatory code that governs gene splicing and expression. CaptureSeq identification of branchpoint nucleotides
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Project description:Both canonical and alternative splicing of RNAs is governed by intronic sequence elements and produces transient lariat structures fastened by branch-points within introns. To map precisely the location of branch-points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes, and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon skipping branch-points. LaSSO thus provides an effective, accurate and unbiased approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated to RNA-seq pipelines. Interrogation of the S. pombe transcriptome using rRNA depleted strand specific RNA sequencing (Illumina HiSeq 2000) in wild type and dbr1.M-NM-^T cultures. A total of 4 samples were analyzed: two biological repeates of wild-type strain and two biological repeats of dbr1.M-NM-^T
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