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.
","abstract_html":"<p>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, <i>in vitro</i> splicing of these introns requires high ionic strength and elevated temperatures. <i>In vivo</i>, 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. </p> <p> 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 <i>in vitro</i>. 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. </p> <p> 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 Mg<sup>2+</sup> induced conformational change, which may be involved in spliceosomal activation <i>in vivo</i>. 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.</p> <p> 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 Mg<sup>2+</sup> as a divalent metal ion. Our FRET data clearly show a Mg<sup>2+</sup> 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 <i>in vivo</i>.</p>","abstract_has_math":false,"creators":["Karunatilaka, Krishanthi Sanjeewani"],"institution":null,"degree_name":"Ph.D.","degree_level":"Open Access Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["David Rueda"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-01-01T08:00:00Z","date_published":"2011-01-01T08:00:00Z","updated_at":"2026-07-24T05:58:49Z","subjects":["Group II introns","Human U2-U6 snRNAs","Mss116","RNA splicing","Single-molecule FRET","Spliceosome","Biochemistry","Biophysics","Molecular Biology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.wayne.edu/oa_dissertations/243","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["David Rueda"]},{"key":"dc:creator","label":"Author","values":["Karunatilaka, Krishanthi Sanjeewani"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2011-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Open Access Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Group II introns","Human U2-U6 snRNAs","Mss116","RNA splicing","Single-molecule FRET","Spliceosome","Biochemistry","Biophysics","Molecular Biology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.wayne.edu/oa_dissertations/243"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Characterization of splicing mechanisms by single-molecule fluorescence"]}]}],"canonical_facts":{"dc:contributor":["David Rueda"],"dc:creator":["Karunatilaka, Krishanthi Sanjeewani"],"dc:date.available":["2011-01-01T08:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.wayne.edu/oa_dissertations/243"],"dc:subject":["Group II introns","Human U2-U6 snRNAs","Mss116","RNA splicing","Single-molecule FRET","Spliceosome","Biochemistry","Biophysics","Molecular Biology"],"dc:title":["Characterization of splicing mechanisms by single-molecule fluorescence"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Open Access Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T05:58:49Z"}