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Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known.
In order to elucidate the mechanism of protein-mediated splicing of group II introns, we have studied the folding dynamics of the D135 ribozyme, a minimal active form of the yeast ai5γ group II intron, in the presence of its natural cofactor, the DEAD-box protein Mss116, using single-molecule fluorescence. Consistent with folding studies at very high magnesium concentrations, our single-molecule data show that Mss116 can promote the folding of group II introns under near physiological conditions in vitro. Furthermore, smFRET data indicate that the Mss116-mediated group II intron folding pathway is a multi-step process that consists of both ATP-independent and ATP-dependent steps.
Structurally and mechanistically group II introns are similar to spliceosome-catalyzed pre-mRNA splicing. Out of five snRNAs, only the highly conserved U2 and U6 snRNAs are required in both steps of RNA splicing. The U2-U6 snRNA complex forms the active site of the spliceosome and has been shown to undergo splicing-related catalysis in the absence of proteins. Single-molecule studies of yeast U2-U6 snRNAs show a Mg2+ induced conformational change, which may be involved in spliceosomal activation in vivo. In contrast to yeast, human U2 and U6 snRNAs contain a large number of post-transcriptional modifications. Recent studies have shown these modifications make human snRNAs more stable than that of yeast indicating a possibility of having different spliceosomal activation states.
In order to understand and compare the catalytic mechanisms, we used single-molecule florescence to characterize the conformational changes of human U2-U6 complex in the presence and absence of modifications using Mg2+ as a divalent metal ion. Our FRET data clearly show a Mg2+ induced conformational change with three FRET states. Based on smFRET data, we propose a minimal two-step folding pathway for human snRNAs similar to yeast. Although unmodified snRNAs exhibit similar folding dynamics as yeast, modified bases destabilize the low FRET state of the U2-U6 complex. However, comparison of FRET and UV melting data suggests modified bases may be involved in protein recognition and/or early assembly of the spliceosome rather than direct stabilization of RNA structures in vivo.
Karunatilaka, Krishanthi Sanjeewani, "Characterization of splicing mechanisms by single-molecule fluorescence" (2011). Wayne State University Dissertations. Paper 243.