Magister Scientiae - MSc (Biochemistry) / Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes. Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves. / South Africa

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Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vii, 91 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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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.

This study seeks to address one of the simplest questions that can be asked about an interconnected system; what happens to one process in the absence of the other process? This is a more difficult task than it would appear at first, due to the absence of small molecule inhibitors that can inhibit splicing globally in yeast cells. The first results chapter describes the adaptation of a system called the auxin induced degron (AID) to the task of inhibiting pre-mRNA splicing. This system appears to have several advantages over previous methods of inhibiting splicing and has many potential applications. Another hurdle to understanding what happens to transcription in the absence of splicing is the differential stability of pre-mRNA versus mRNA. At steady state the vast majority of transcripts of a specified gene will be mRNA transcripts. This means that even if you could rapidly inhibit splicing it would be a long time before all the pre-existing mRNA would turn-over. If you waited until specified mRNAs turned over it is likely that the cells would be very sick making it difficult to separate primary and secondary effects. The second results chapter shows the use of a metabolic labelling technique using a uracil analogue called 4-thiouracil (4SU). 4SU is added for an extremely short amount of time (1.5 min, 2.5 min, and 5 min) and the RNA produced during the labelling time is isolated by affinity purification. This allows us to study the kinetics of pre-mRNA splicing in wild-type cells and to seek correlations between splicing kinetics and gene architecture. The third results chapter combines the methods used in the previous two chapters to give a new technique called AID4U-seq. AID4U-seq allows for rapid inhibition of splicing and then the ability to isolate only the transcripts that were created after this inhibition came into effect. This should allow for examination of the primary consequences of blocking pre-mRNA splicing at multiple stages during spliceosome assembly. Additionally AID4U-seq is immediately applicable to the study of other areas of RNA processing. Defining the effects on the transcriptome of inhibiting splicing at multiple stages of assembly is an ambitious aim likely to require many more years of research. Therefore this thesis chiefly seeks to illustrate a novel strategy to begin dissecting a complex issue in which splicing, transcription, degradation and the post-transcriptional modification of histones are all likely to have roles.

<p>This review article presents the splicing process during messenger RNA maturation and how it is regulated by different <em>Cis</em>-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism <em>Arabidopsis thaliana</em> is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.</p>

Nucleic acids are an emerging class of therapeutics with the capacity to repair both DNA and RNA mutations in clinically relevant targets. We have used two approaches, mobile group II introns and Spliceosome Mediated RNA Trans-splicing (SMaRT), to correct β-globin mutations at the DNA and RNA levels respectively. We show that the group II intron inserts site-specifically into its DNA target, even when similar targets are available. Experiments transitioning this therapeutic into mammalian cell systems are then described. We also illustrate how SMaRT RNA repair can be used to correct β-globin mutations involved in sickle cell disease and some forms of β- thalassemia. We uncovered diverse repair efficiencies when targeting sickle cell versus β- thalassemia transcripts in mammalian cells. Possible reasons for this and how it might direct target choice for the SMaRT therapeutic approach are both discussed. The therapeutic molecule in SMaRT, a Pre-Trans-splicing Molecule or PTM, is also delivered via lentivirus to erythrocyte precursors cultured from the peripheral blood of sickle cell patients. Preliminary results from these experiments are discussed. / Dissertation

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.