Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

David Rueda

Abstract

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.

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