Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor(s)

Nancy L. Greenbaum

Subject Categories

Biochemistry | Biophysics | Chemistry

Keywords

conformation, snRNA, Spliceosome, structure probing, U2-U6

Abstract

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several specifically bound Mg2+ binding sites in vitro. The overall goal of this research is to characterize the conformational changes of the human U2-U6 snRNA complex upon addition of Mg2+. In order to pursue this question, we attempted to characterize the lowest energy structure of the complex in the absence of spliceosomal proteins using a combination of biophysical and biochemical techniques in the solution state. We first used enzymatic structure probing to evaluate the secondary structural fold of protein-free human U2-U6 snRNA complex. Cleavage patterns resulting from probing reactions were consistent with formation of four stem regions surrounding the junction, therefore favoring the four-helix model consistent with previous results of in vivo studies of the human U2-U6 snRNA complex. However, 19F NMR studies from our laboratory also identified a lesser fraction (up to 14%) of a three- helix conformation. Upon addition of up to 100 mM Mg2+, a slight increase in cleavage by enzymes specific for both single-stranded and double-stranded regions was observed at the junction region, suggesting that this region is becoming more accessible, perhaps because of an increase in the fraction of the three-helix conformation. Analytical ultracentrifugation studies revealed that the Stokes radius of the RNA complex decreased slightly from 31.3 Ã? to 27.9 Ã? in the presence of 100 mM Mg2+, suggesting a slight compaction of the tertiary structure in the presence of divalent metal ions. Hydroxyl radical footprinting experiments on this complex showed signs of increased protection in some areas near and more distant from the junction upon addition of Mg2+, suggesting a change in three-dimensional conformation. Therefore, it appears that Mg2+ induces a small three-dimensional conformational change on human U2-U6 snRNA complex. In order to build a three-dimensional model for the four-helix conformation, we designed a mutant that favors the formation of four-helix conformation and performed SAXS experiments on it. The preliminary SAXS studies suggest that the human U2-U6 snRNA complex and the mutant complex may also be amenable to further study by SAXS. These results act as a good starting point to characterize further the overall global conformation of human U2-U6 snRNA complex and effects of spliceosomal proteins on it.

 
 

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