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In addressing the pre-mRNA substrate, the splicing machinery requires rearrangement of multiple RNA and protein components. The classical model of spliceosome formation begins with the U1 snRNA recognition of the 5" splice site and U2 snRNP interaction with the branch point. This process is followed by the engagement of a pre-assembled U4/U6·U5 tri-snRNP to form the A2-1 complex. The spliceosome is subsequently activated through a number of structural rearrangements. Among these is the unwinding of the U4/U6 intermolecular helix by the tri-snRNP component Brr2p. While numerous protein components of the tri-snRNP have been identified, the function of many of these remain unknown. The nonessential Snu66p (U4/U6·U5-110K in humans) stably associates only with the U4/U6·U5 tri-snRNP while the similarly nonessential Snu40p (U5-52K in humans) associates exclusively with the U5 snRNP. To understand why two non-essential pre-mRNA splicing factors have been so well conserved through great evolutionary distances, we examined their roles in the assembly and function of the tri-snRNP. Removal of SNU40 alone does not affect snRNP levels, however deletion of SNU66 results in reduced levels of tri-snRNP. The U4/U6·U5 snRNPs in [Delta]snu66 cells are resistant to the ATP-dependent U4/U6 unwinding by Brr2p, and profound U4/U6 accumulation occurs at reduced temperatures. Remarkably, subsequent removal of SNU40 in a [Delta]snu66 strain bypasses the tri-snRNP formation defect while unwinding of U4/U6 remains defective. Additional investigation revealed that Prp6p, another tri-snRNP protein, is destabilized from the complex. Based upon this data in total, I present a model in which Snu40p and Snu66p interact sequentially with Prp6p to maintain directionality for proper biogenesis of the tri-snRNP. Further, the U4/U6 unwinding defect of the double mutant should theoretically arrest the A2-1 spliceosome. Indeed, native gel analysis confirms the buildup of a large complex later determined to be A2-1. I have purified this complex, functionally tested its catalytic viability, and identified its components via mass spectrometry. This is the first full characterization of the A2-1 precatalytic spliceosome complex in Saccharomyces cerevisiae. / text

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Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisor: Alan M. Lambowitz. Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: George Georgiou. Vita. Includes bibliographical references.

No description available.

The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy