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Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Lars Feuk's group)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-6085-6749
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 13, 99- p.Article in journal (Refereed) Published
Abstract [en]

Background: The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression. Results: We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics. Conclusion: Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.

Place, publisher, year, edition, pages
2013. Vol. 13, 99- p.
Keyword [en]
Cuyoplasmic RNA, Nuclear RNA, RNA sequencing, mRNA
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-209270DOI: 10.1186/1472-6750-13-99ISI: 000327430800001OAI: oai:DiVA.org:uu-209270DiVA: diva2:656557
Available from: 2013-10-16 Created: 2013-10-16 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Genome-wide Characterization of RNA Expression and Processing
Open this publication in new window or tab >>Genome-wide Characterization of RNA Expression and Processing
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out.

In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis.

The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 939
Keyword
RNA sequencing, RNA splicing, RNA processing, Gene expression
National Category
Medical Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-209390 (URN)978-91-554-8784-3 (ISBN)
Public defence
2013-11-29, Rudbeck Salen, Dag Hammarskjölds väg 20,, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-10-18 Last updated: 2014-01-23

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Zaghlool, AmmarAmeur, AdamNyberg, LinneaHalvardson, JonatanGrabherr, ManfredCavelier, LuciaFeuk, Lars

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Zaghlool, AmmarAmeur, AdamNyberg, LinneaHalvardson, JonatanGrabherr, ManfredCavelier, LuciaFeuk, Lars
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Department of Immunology, Genetics and PathologyScience for Life Laboratory, SciLifeLabDepartment of Medical Biochemistry and Microbiology
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