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Genome-wide Characterization of RNA Expression and Processing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Lars Feuk's group)
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 [en]
RNA sequencing, RNA splicing, RNA processing, Gene expression
National Category
Medical Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:uu:diva-209390ISBN: 978-91-554-8784-3 (print)OAI: oai:DiVA.org:uu-209390DiVA: diva2:657213
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
List of papers
1. Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain
Open this publication in new window or tab >>Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain
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2011 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 18, no 12, 1435-1440 p.Article in journal (Refereed) Published
Abstract [en]

Transcriptome sequencing allows for analysis of mature RNAs at base pair resolution. Here we show that RNA-seq can also be used for studying nascent RNAs undergoing transcription. We sequenced total RNA from human brain and liver and found a large fraction of reads (up to 40%) within introns. Intronic RNAs were abundant in brain tissue, particularly for genes involved in axonal growth and synaptic transmission. Moreover, we detected significant differences in intronic RNA levels between fetal and adult brains. We show that the pattern of intronic sequence read coverage is explained by nascent transcription in combination with co-transcriptional splicing. Further analysis of co-transcriptional splicing indicates a correlation between slowly removed introns and alternative splicing. Our data show that sequencing of total RNA provides unique insight into the transcriptional processes in the cell, with particular importance for normal brain development.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-166861 (URN)10.1038/nsmb.2143 (DOI)000298011600036 ()
Note

De två sista författarna delar sistaförfattarskapet

Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2017-12-08Bibliographically approved
2. Exome RNA sequencing reveals rare and novel alternative transcripts
Open this publication in new window or tab >>Exome RNA sequencing reveals rare and novel alternative transcripts
2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 1, e6- p.Article in journal (Refereed) Published
Abstract [en]

RNA sequencing has become an important method to perform hypothesis-free characterization of global gene expression. One of the limitations of RNA sequencing is that most sequence reads represent highly expressed transcripts, whereas low level transcripts are challenging to detect. To combine the benefits of traditional expression arrays with the advantages of RNA sequencing, we have used whole exome enrichment prior to sequencing of total RNA. We show that whole exome capture can be successfully applied to cDNA to study the transcriptional landscape in human tissues. By introducing the exome enrichment step, we are able to identify transcripts present at very low levels, which are below the level of detection in conventional RNA sequencing. Although the enrichment increases the ability to detect presence of transcripts, it also lowers the accuracy of quantification of expression levels. Our results yield a large number of novel exons and splice isoforms, suggesting that conventional RNA sequencing methods only detect a small fraction of the full transcript diversity. We propose that whole exome enrichment of RNA is a suitable strategy for genome-wide discovery of novel transcripts, alternative splice variants and fusion genes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-180350 (URN)10.1093/nar/gks816 (DOI)000312889900006 ()22941640 (PubMedID)
Available from: 2012-09-04 Created: 2012-09-04 Last updated: 2017-12-07Bibliographically approved
3. Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues
Open this publication in new window or tab >>Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues
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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.

Keyword
Cuyoplasmic RNA, Nuclear RNA, RNA sequencing, mRNA
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-209270 (URN)10.1186/1472-6750-13-99 (DOI)000327430800001 ()
Available from: 2013-10-16 Created: 2013-10-16 Last updated: 2017-12-06Bibliographically approved
4. Mutation in the chromatin-remodeling factor BAZ1A is associated with intellectual disability
Open this publication in new window or tab >>Mutation in the chromatin-remodeling factor BAZ1A is associated with intellectual disability
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Exome sequencing has led to the identification of mutations in several genes involved in chromatin remodeling in syndromic forms of intellectual disability. Here, we used exome sequencing to identify a single non-synonymous de novo mutation in BAZ1A, encoding the ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained intellectual disability. ACF1 has been previously reported to bind to the promoter of vitamin D receptor (VDR) regulated genes and suppress their expression in the absence of vitamin D. We found that the mutation in BAZ1A affects the expression of many genes, mainly involved in extra cellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlates with the clinical diagnosis of the patient. We therefore propose that BAZ1A represents yet another chromatin remodeling gene involved in causing an intellectual disability syndrome.

National Category
Other Medical Sciences
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-209275 (URN)
Available from: 2013-10-16 Created: 2013-10-16 Last updated: 2014-01-23Bibliographically approved

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