title: Conformational dynamics of stem II of the U2 snRNA.
abstract: The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg(2+) and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg(2+) and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg(2+) may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg(2+) or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg(2+) toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg(2+)-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Rodgers
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: U Sandy
authors.initials: US
authors.lastname: Tretbar
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dehaven
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Amir A
authors.initials: AA
authors.lastname: Alwan
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: George
authors.initials: G
authors.lastname: Luo
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Mast
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C114067:CUS2 protein, S cerevisiae; D012322:RNA Precursors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA
keywords: RNA; dynamics; single-molecule FRET; snRNP; spliceosome