Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging
from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5
and U6) involved in substrate positioning and catalysis. It has been postulated, but never
shown experimentally, that the U2-U6 complex adopts at least two conformations that
reflect different activation states. We have used single-molecule fluorescence to probe the
structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and
mutants of highly conserved regions of U2-U6. Our data show the presence of at least
three distinct conformations in equilibrium. The minimal folding pathway consists of a
two-step process with an obligatory intermediate. The first step is strongly magnesium
dependent, and we provide evidence suggesting that the second step corresponds to the
formation of the genetically conserved helix IB. Site-specific mutations in the highly
conserved AGC triad and the U80 base in U6 suggest that the observed conformational
dynamics correlate with residues that have an important role in splicing. We also report
the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a
corresponding set of base triple interactions discovered in the recent published crystal
structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base
triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the
ones found in crystal structure of the self-splicing group II intron given the extensive
similarities between the spliceosome and the group II intron. Our data agree very well
with the hypothesis. There are a large number of proteins in the spliceosome that play
vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural
rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing
that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.
This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits
the binding of Mg2+ to U2/U6.
","abstract_html":"<p>Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging</p> <p>from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5</p> <p>and U6) involved in substrate positioning and catalysis. It has been postulated, but never</p> <p>shown experimentally, that the U2-U6 complex adopts at least two conformations that</p> <p>reflect different activation states. We have used single-molecule fluorescence to probe the</p> <p>structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and</p> <p>mutants of highly conserved regions of U2-U6. Our data show the presence of at least</p> <p>three distinct conformations in equilibrium. The minimal folding pathway consists of a</p> <p>two-step process with an obligatory intermediate. The first step is strongly magnesium</p> <p>dependent, and we provide evidence suggesting that the second step corresponds to the</p> <p>formation of the genetically conserved helix IB. Site-specific mutations in the highly</p> <p>conserved AGC triad and the U80 base in U6 suggest that the observed conformational</p> <p>dynamics correlate with residues that have an important role in splicing. We also report</p> <p>the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a</p> <p>corresponding set of base triple interactions discovered in the recent published crystal</p> <p>structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base</p> <p>triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the</p> <p>ones found in crystal structure of the self-splicing group II intron given the extensive</p> <p>similarities between the spliceosome and the group II intron. Our data agree very well</p> <p>with the hypothesis. There are a large number of proteins in the spliceosome that play</p> <p>vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural</p> <p>rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing</p> <p>that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.</p> <p>This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits</p> <p>the binding of Mg2+ to U2/U6.</p>","abstract_has_math":false,"creators":["Guo, Zhuojun"],"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":2010,"date_issued":"2010-01-01T08:00:00Z","date_published":"2010-01-01T08:00:00Z","updated_at":"2026-07-24T05:58:42Z","subjects":["single molecule","splicing","U2-U6","Biophysics"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.wayne.edu/oa_dissertations/163","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":["Guo, Zhuojun"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2011-01-04T08:00:00Z"]},{"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":["single molecule","splicing","U2-U6","Biophysics"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.wayne.edu/oa_dissertations/163"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging
from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5
and U6) involved in substrate positioning and catalysis. It has been postulated, but never
shown experimentally, that the U2-U6 complex adopts at least two conformations that
reflect different activation states. We have used single-molecule fluorescence to probe the
structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and
mutants of highly conserved regions of U2-U6. Our data show the presence of at least
three distinct conformations in equilibrium. The minimal folding pathway consists of a
two-step process with an obligatory intermediate. The first step is strongly magnesium
dependent, and we provide evidence suggesting that the second step corresponds to the
formation of the genetically conserved helix IB. Site-specific mutations in the highly
conserved AGC triad and the U80 base in U6 suggest that the observed conformational
dynamics correlate with residues that have an important role in splicing. We also report
the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a
corresponding set of base triple interactions discovered in the recent published crystal
structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base
triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the
ones found in crystal structure of the self-splicing group II intron given the extensive
similarities between the spliceosome and the group II intron. Our data agree very well
with the hypothesis. There are a large number of proteins in the spliceosome that play
vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural
rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing
that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.
This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits
the binding of Mg2+ to U2/U6.
"]},{"key":"dc:title","label":"Title","values":["Single Molecule Studies Of Spliceosomal Snrnas U2-U6"]}]}],"canonical_facts":{"dc:contributor":["David Rueda"],"dc:creator":["Guo, Zhuojun"],"dc:date.available":["2011-01-04T08:00:00Z"],"dc:description.abstract":["Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging
from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5
and U6) involved in substrate positioning and catalysis. It has been postulated, but never
shown experimentally, that the U2-U6 complex adopts at least two conformations that
reflect different activation states. We have used single-molecule fluorescence to probe the
structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and
mutants of highly conserved regions of U2-U6. Our data show the presence of at least
three distinct conformations in equilibrium. The minimal folding pathway consists of a
two-step process with an obligatory intermediate. The first step is strongly magnesium
dependent, and we provide evidence suggesting that the second step corresponds to the
formation of the genetically conserved helix IB. Site-specific mutations in the highly
conserved AGC triad and the U80 base in U6 suggest that the observed conformational
dynamics correlate with residues that have an important role in splicing. We also report
the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a
corresponding set of base triple interactions discovered in the recent published crystal
structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base
triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the
ones found in crystal structure of the self-splicing group II intron given the extensive
similarities between the spliceosome and the group II intron. Our data agree very well
with the hypothesis. There are a large number of proteins in the spliceosome that play
vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural
rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing
that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.
This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits
the binding of Mg2+ to U2/U6.
"],"dc:identifier":["https://digitalcommons.wayne.edu/oa_dissertations/163"],"dc:subject":["single molecule","splicing","U2-U6","Biophysics"],"dc:title":["Single Molecule Studies Of Spliceosomal Snrnas U2-U6"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Open Access Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T05:58:42Z"}