Date of Degree

6-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor(s)

Nancy L. Greenbaum

Committee Members

Nancy L. Greenbaum

Ruth Stark

Ranajeet Ghose

Akira Kawamura

Subject Categories

Biochemistry | Biophysics | Other Chemistry | Structural Biology

Keywords

Protein, RNA, NMR, EMSA, Spliceosome, p14

Abstract

Newly transcribed precursor messenger RNA (pre-mRNA) molecules contain coding sequences (exons) interspersed with non-coding intervening sequences (introns). These introns must be removed in order to generate a continuous coding sequence prior to translation of the message into protein. The mechanism through which these introns are removed is known as pre-mRNA splicing, a two-step reaction catalyzed be a large macromolecular machine, the spliceosome, located in the nucleus of eukaryotic cells. The spliceosome is a protein-directed ribozyme composed of small nuclear RNAs (snRNA) and hundreds of proteins that assemble in a very dynamic process. One of these snRNAs, the U2 snRNA, is an important component of the human spliceosomal catalytic core that pairs with the intron through the branch site interacting region. These interactions are stabilized by the presence of several protein splicing factors. One splicing factor, p14, is the only protein shown to interact directly with the branch site in the fully assembled spliceosome. In this research we have used electrophoretic mobility shift assays (EMSA) and nuclear magnetic resonance (NMR) under non-denaturing conditions to establish the structural and or functional role of this splicing factor. Our EMSA results show that p14, which contains a RNA recognition motif (RRM), binds duplex RNA representing the branch site helix (yBP) with weak affinity (KD in the range of 200-400 mM). However, p14 also binds single-stranded (ss) RNA and even a non-related double-stranded (ds) DNA; therefore, any binding appears to be nonspecific for sequence or pairing status. The p14 protein also interacted with a fragment representing the SF3b155 protein, a natural binding partner in the spliceosome forming a stable and strong complex. Our NMR studies show that ten cross-peaks of 15N-labeled p14 were perturbed upon interaction with yBP. Calculations of the magnitude of the chemical shift changes upon titration of RNA into the protein solution suggested KD values of ~150 mM. However, perturbations in the presence of ss intron, ssU2 snRNA or dsDNA of a different sequence are similar to those with the branch site duplex, further supporting the finding from EMSA that interaction is non-specific for sequence, pairing status, or even nucleic acid. In the presence of the SF3b155 fragment, most of the p14 cross-peaks were perturbed consistent with extensive protein-protein contact. In this case, addition of the RNA duplex resulted in shifts in only a subset of the cross-peaks in p14 seen in the absence of SF3b155 but with similar affinity. Our NMR data imply that p14 interacts with RNA through very electropositive regions located in its RNP2 motif and a β-loop, with or without the SF3b155 fragment. However, no residues on β3, the RNP1 motif that usually interacts with ssRNA in RRM proteins, showed significant perturbation. Affinity, as determined by NMR titration, for yBP and an RNA duplex without the branch site (yBPΔA) were very similar. However, the overall magnitude of chemical shift perturbations was larger for yBP than for yBPΔA, which we speculate is related to the highly negative surface potential of yBP in the major groove. Taken together, p14 interacts with the branch site RNA and the binding appears to be of an electrostatic nature between the electropositive patch of RNP2 and the negative backbone of the RNA. Thus, we speculate that the role of p14 in human spliceosome is an electrostatic spacer as a cofactor of SF3b155 to screen backbone charges of the branch site RNA during spliceosome assembly to protect branch site from premature chemical activity prior to formation of the spliceosome’s active site.

 
 

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