Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor

Nancy Greenbaum

Committee Members

Kevin Ryan

Yujia Xu

Subject Categories

Other Biochemistry, Biophysics, and Structural Biology | Structural Biology

Keywords

U2-U6 snRNA, junction, spliceosome, time-resolved FRET, EMSA, RBM22, CWC2

Abstract

Splicing of precursor messenger RNA is an essential process in eukaryotes in which the non-coding regions (introns) are removed and coding regions (exons) ligated together to form a mature mRNA. This process is catalyzed by a multi-mega Dalton ribonucleoprotein complex called the spliceosome, which is assembled from five small nuclear ribonucleoproteins (snRNP) in the form of RNA-protein complexes (U1, U2, U4, U5 and U6) and hundreds of proteins. U2 and U6 small nuclear (sn)RNAs are the only snRNAs directly implicated in catalyzing the splicing of pre-mRNA, but assembly and rearrangement steps prior to catalysis require numerous proteins. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations in equilibrium, characterized by four and three helices surrounding a central junction. The four-helix conformer is strongly favored in the in vitro protein-free state, but the three-helix conformer predominates in spliceosomes. To analyze the role of the central junction in positioning elements forming the active site, we derived three-dimensional models of the two conformations from distances measured between fluorophores at selected locations in constructs representing the protein-free human U2-U6 snRNA complex by time-resolved fluorescence resonance energy transfer. Data describing four angles in the four-helix conformer suggest tetrahedral geometry; addition of Mg2+ results in shortening of the distances between neighboring helices, indicating compaction of the complex around the junction. In contrast, the three-helix conformer shows a closer approach between helices bearing critical elements, but addition of Mg2+ widens the distance between them and enhances the fraction of the three-helix conformer. Thus, in neither conformer are the critical helices positioned to favor the proposed triplex interaction suggesting the obligation for other components, spliceosomal proteins, to facilitate and stabilize the catalytically active conformation. We showed that incubation with the NineTeen Complex (NTC)-related protein RBM22, which has been implicated in the remodeling of the U2-U6 snRNA complex to render it catalytically active, favors the formation of the three-helix conformer as seen in cryo-EM images of spliceosome. Binding analysis of interaction between yU2-U6 snRNA and a partial homologue of RBM22 in yeast, Cwc2, reveals a specific interaction between them, indicating a specific conformation of the active site stabilized by the protein. These data suggest that although the central junction assumes a significant role in orienting helices, spliceosomal proteins and Mg2+ facilitate formation of the catalytically active conformer.

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