Thesis (Ph.D.)--Washington State University. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Alan M. Lambowitz. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Florida State University, 2006. / Advisor: Nancy L. Greenbaum, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Jan. 2, 2007). Document formatted into pages; contains xix, 173 pages. Includes bibliographical references.

DNA sequence analysis involves precise discrimination of two of the sequence's most important components: exons and introns. Exons encode the proteins that are responsible for almost all the functions in a living organism. Introns interrupt the sequence coding for a protein and must be removed from primary RNA transcripts before translation to protein can occur. A pattern recognition technique called Finite Induction (FI) is utilized to study the language of exons and introns. FI is especially suited for analyzing and classifying large amounts of data representing sequences of interest. It requires no biological information and employs no statistical functions. Finite Induction is applied to the exon and intron components of DNA by building a collection of rules based upon what it finds in the sequences it examines. It then attempts to match the known rule patterns with new rules formed as a result of analyzing a new sequence. A high number of matches predict a probable close relationship between the two sequences; a low number of matches signifies a large amount of difference between the two. This research demonstrates FI to be a viable tool for measurement when known patterns are available for the formation of rule sets.

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

In eukaryotes the coding regions of most genes are interrupted by introns that must be removed by splicing to form a coding mRNA. However, while the splicing mechanism has received a lot of attention, much less is known about the metabolism of introns. This is partly due to the difficulties in studying introns as both aberrantly spliced transcripts and spliced introns are rapidly degraded. In this study, I have analysed intron metabolism in two respects: first I have investigated how introns are degraded following the completion of splicing. Second, I investigate the fate of transcripts, in which introns are retained due to splicing failure. In order to study the degradation of introns following splicing, I performed siRNA mediated knock down of the debrancing enzyme (Dbr1). Following splicing, introns are present in a circular lariat structure and Dbr1 is the enzyme thought to be responsible for opening this. Indeed, I found that knockdown of Dbr1 increased the amount of stabilised introns. Interestingly, introns were found to be stabilised in the cytoplasm and not in the nucleus as expected, even though immunofluoresence showed that Dbr1 is clearly nuclear. However, western blot analysis localised Dbr1 in the cytoplasm. Further investigation showed widely used methods to separate nuclear and cytoplasmic fractions are prone to generating artefacts which result in nucleoplasmic proteins delocalised to the cytoplasm. This finding may prevent future misinterpretation of data obtained by these methods. To investigate splicing failure, it was necessary to generated a sufficiently large pool of unspliced transcripts. To do this I used antisense morpholinos (AMOs) that bind to specific snRNAs (small nuclear RNAs). They are designed to block interaction surfaces that are important for splicing. Using this approach, I investigated the localisation of RNA transcripts and selected RNA processing and degradation factors in normal conditions and where splicing was inhibited. When splicing is inhibited I found splicing factors and unspliced, polyadenylated RNA localising to nuclear, splicing speckle marker SC35 positive speckles. I further discovered that for RNA to localise to nuclear speckles, polyadenylation and RNA cleavage are essential, indicating that SC-35 speckles might sequester unspliced transcripts preventing translation of potentially harmful transcripts. These transcripts remain functional however, and can be spliced where functional spliceosomes can be assembled.

Not available / text

Not available / text

DExD/H-box proteins are a diverse class of proteins that are implicated in RNA and RNP remodeling. They have sequence homology to DNA helicases and share conserved ATPase domains, suggesting that they use the energy of ATP binding and hydrolysis to mediate conformational rearrangements in RNAs. In the past, the action of DExD/H-box proteins has been characterized primarily on simple model substrates such as small RNA duplexes. It is not known how DExD/H-box proteins manipulate structured RNA, what determines target specificity and what molecular events follow their action. Here, using the well-characterized Tetrahymena group I intron ribozyme, I performed kinetic and thermodynamic studies to understand the mechanism of CYT-19 and related DExD/Hbox proteins. CYT-19 has been shown previously to facilitate the folding of several group I and group II introns. I demonstrated that CYT-19 acts as a chaperone, accelerating the re-folding of a long-lived misfolded species of the Tetrahymena group I ribozyme to its native state. Further characterization of this reaction gave insights into how CYT-19 achieves this action; CYT-19 partially unfolds the misfolded ribozyme and allows it to fold again along the same pathway that exists in the absence of CYT-19. In addition to acting on the misfolded state, CYT-19 also acts on the native state, but this action is largely obscured under stabilizing conditions for the native state because the action is inefficient under such conditions. However, under conditions where the native state is destabilized, the native ribozyme was indeed shown to be partially unfolded by CYT-19. By acting on either species, CYT-19 sets up a steady state of unfolding, and the distribution is shifted from equilibrium to kinetic control, increasing the relative populations of conformations that are kinetically preferred during folding. The efficiency of action seems to correlate with the stability of the ribozyme. These activities are not restricted to CYT-19; the DExD/H-box proteins Mss116p and Ded1 were demonstrated to possess similar activities. Together, these studies give important insights into the mechanisms of action for this ubiquitous class of proteins and have implications for all structured RNAs in cells. / text

Group II introns insert site-specifically into DNA target sites through a process termed retrohoming. They consist of a structured, catalytically active intron RNA and its encoded protein. The protein contains several domains, including a reverse transcriptase domain and a DNA endonuclease domain used for bottom-strand cleavage. Recently, the thermophile Thermosynechococcus elongatus BP-1 was found to contain eight functional group II intron-encoded proteins. The proteins are thermostable and active at temperatures up to 65°C. The intron-encoded protein, TeI4c displays the greatest reverse transcriptase activity of these eight proteins, as well as high fidelity and processivity; ideal qualities for a commercial reverse transcriptase. This work explores the possibility of using TeI4c for biotechnology applications, and specifically examines the C-terminal endonuclease domain of TeI4c and its effect on reverse transcription. Additionally, this work investigates the retrohoming activity of a TeI4c truncation that deletes the endonuclease domain. / text