Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms. / text

published_or_final_version / Medicine / Master / Master of Philosophy

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-127).

The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail. / text

No description available.

Thesis (M.Phil.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 65-71) Also available in print.