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Construction and deep sequencing of cDNA libraries representing medium-sized RNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Department of Biomolecular Engineering, University of California, Santa Cruz.
Max Planck Institute for Evolutionary Anthropology.
Max Planck Institute for Evolutionary Anthropology.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transcriptome analysis has recently become feasible with the application of RNA-seq, the high-throughput sequencing analysis of RNAs through cDNA synthesis. Current transcriptome analysis approaches are limited to analysis of long (>200 nts) or short (~20 nts) RNAs. Here we describe an approach that can be used for the analysis of the medium-sized (~200 nts) RNA transcriptome, which reveals the full-length sequence of RNAs at the single molecule level. Our approach can easily be adapted to generate libraries that are suitable for sequencing by several of the currently used next-generation platforms.

Keyword [en]
high-throughput sequencing, 454, snRNA, cDNA library
National Category
Natural Sciences Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
URN: urn:nbn:se:uu:diva-172422OAI: oai:DiVA.org:uu-172422DiVA: diva2:514629
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2012-08-01
In thesis
1. Understanding the Noise: Spliceosomal snRNA Profiling
Open this publication in new window or tab >>Understanding the Noise: Spliceosomal snRNA Profiling
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The concept of the gene has been constantly challenged by new discoveries in the life sciences. Recent challenging observations include the high frequency of alternative splicing events and the common transcription of non-protein-coding-RNAs (ncRNAs) from the genome. The latter has long been considered noise in biological systems. Multiple lines of evidence from genomic studies indicate that alternative splicing and ncRNA play important roles in expanding proteome diversity in eukaryotes. Here, the aim is to find the link between alternative splicing and ncRNAs by studying the expression profile of the spliceosomal snRNAs (U snRNA).

Spliceosomal snRNAs are essential for pre-mRNA splicing in eukaryotes. They participate in splice site selection, recruitment of protein factors and catalyzing the splicing reaction. Because of this, both the abundance and diversity of U snRNAs were expected to be large. In our study we deeply analyzed the U snRNA population in primates using a combination of bioinformatical, biochemical and high throughput sequencing approaches. This transcriptome profiling has revealed that human, chimpanzee and rhesus have similar U snRNA populations, i.e. the vast majority of U snRNAs originate from few well-defined gene loci and the heterogeneity observed in U snRNA populations was largely due to the presence of SNPs at these loci. It seems that the gene loci that could potentially encode a significantly heterogeneous population of U snRNAs are mostly silent. Only few minority transcripts were detected in our study, and among them three U1-like snRNAs might play a role in the regulation of alternative splicing by recognizing non-canonical splicing sites.

Mutations of U snRNA have been shown to impact the splicing process. Therefore, our study provides a reference to study the biological significance of SNPs in U snRNA genes and their association with diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 924
Keyword
ncRNA, snRNA, U1, splice site, alternative splicing, high-throughput sequencing, 454, SNP
National Category
Natural Sciences Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-172403 (URN)978-91-554-8341-8 (ISBN)
Public defence
2012-05-24, B41, Husargatan 3, Biomedicinskt Centrum, Uppsala University, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2012-05-02 Created: 2012-04-10 Last updated: 2012-08-01Bibliographically approved

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CiteExportLink to record
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