Dataset Information

Genome-wide identification of spliced introns using a tiling microarray.

ABSTRACT: The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA from the debranching mutant to the wild-type parent strain, and thus the identification of lariats on a genome-wide scale. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others. In many instances, signals from the tiling array delineated the 5' splice site and branchpoint site, confirming predicted gene structures. Nearly all introns that went undetected are present in mRNAs expressed at low levels. Overall, 97% of the significant probes could be attributed either to spliced introns or to genes up-regulated by deletion of the debranching enzyme. Because the debranching enzyme is conserved among eukaryotes, this approach could be generally applicable for the annotation of eukaryotic genes and the detection of novel and alternative splice forms.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC1832097 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Genome-wide identification of spliced introns using a tiling microarray.

Zhang Zhihong Z Hesselberth Jay R JR Fields Stanley S

Genome research 20070309 4

The prediction of gene models from genome sequence remains an unsolved problem. One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. The excised introns are released in the form of lariats, which must be debranched prior to their turnover. In the yeast Saccharomyces cerevisiae, the absence of the debranching enzyme causes these lariat RNAs to accumulate. This accumulation allows a comparison of tiling array signals of RNA ...[more]

PMID: 17351133

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Project description:BackgroundIt is generally thought that most canonical or non-canonical splicing events involving U2- and U12 spliceosomes occur within nuclear pre-mRNAs. However, the question of whether at least some U12-type splicing occurs in the cytoplasm is still unclear. In recent years next-generation sequencing technologies have revolutionized the field. The "Read-Split-Walk" (RSW) and "Read-Split-Run" (RSR) methods were developed to identify genome-wide non-canonical spliced regions including special events occurring in cytoplasm. As the significant amount of genome/transcriptome data such as, Encyclopedia of DNA Elements (ENCODE) project, have been generated, we have advanced a newer more memory-efficient version of the algorithm, "Read-Split-Fly" (RSF), which can detect non-canonical spliced regions with higher sensitivity and improved speed. The RSF algorithm also outputs the spliced sequences for further downstream biological function analysis.ResultsWe used open access ENCODE project RNA-Seq data to search spliced intron sequences against the U12-type spliced intron sequence database to examine whether some events could occur as potential signatures of U12-type splicing. The check was performed by searching spliced sequences against 5'ss and 3'ss sequences from the well-known orthologous U12-type spliceosomal intron database U12DB. Preliminary results of searching 70 ENCODE samples indicated that the presence of 5'ss with U12-type signature is more frequent than U2-type and prevalent in non-canonical junctions reported by RSF. The selected spliced sequences have also been further studied using miRBase to elucidate their functionality. Preliminary results from 70 samples of ENCODE datasets show that several miRNAs are prevalent in studied ENCODE samples. Two of these are associated with many diseases as suggested in the literature. Specifically, hsa-miR-1273 and hsa-miR-548 are associated with many diseases and cancers.ConclusionsOur RSF pipeline is able to detect many possible junctions (especially those with a high RPKM) with very high overall accuracy and relative high accuracy for novel junctions. We have incorporated useful parameter features into the pipeline such as, handling variable-length read data, and searching spliced sequences for splicing signatures and miRNA events. We suggest RSF, a tool for identifying novel splicing events, is applicable to study a range of diseases across biological systems under different experimental conditions.

Project description:Introns exist not only in coding sequences (CDSs) but also in untranslated regions (UTRs) of a gene. Recent studies in animals and model plants such as Arabidopsis have revealed that the UTR-introns (UIs) are widely presented in most genomes and involved in regulation of gene expression or RNA stability. In the present study, we identified introns at both 5'UTRs (5UIs) and 3'UTRs (3UIs) of sweet orange genes, investigated their size and nucleotide distribution characteristics, and explored the distribution of cis-elements in the UI sequences. Functional category of genes with predicted UIs were further analyzed using GO, KEGG, and PageMan enrichment. In addition, the organ-dependent splicing and abundance of selected UI-containing genes in root, leaf, and stem were experimentally determined. Totally, we identified 825 UI- and 570 3UI-containing transcripts, corresponding to 617 and 469 genes, respectively. Among them, 74 genes contain both 5UI and 3UI. Nucleotide distribution analysis showed that 5UI distribution is biased at both ends of 5'UTR whiles 3UI distribution is biased close to the start site of 3'UTR. Cis- elements analysis revealed that 5UI and 3UI sequences were rich of promoter-enhancing related elements, indicating that they might function in regulating the expression through them. Function enrichment analysis revealed that genes containing 5UI are significantly enriched in the RNA transport pathway. While, genes containing 3UI are significantly enriched in splicesome. Notably, many pentatricopeptide repeat-containing protein genes and the disease resistancegenes were identified to be 3UI-containing. RT-PCR result confirmed the existence of UIs in the eight selected gene transcripts whereas alternative splicing events were found in some of them. Meanwhile, qRT-PCR result showed that UIs were differentially expressed among organs, and significant correlation was found between some genes and their UIs, for example: The expression of VPS28 and its 3UI was significantly negative correlated. This is the first report about the UIs in sweet orange from genome-wide level, which could provide evidence for further understanding of the role of UIs in gene expression regulation.

Dataset Information

Genome-wide identification of spliced introns using a tiling microarray.

Publications

Genome-wide identification of spliced introns using a tiling microarray.

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