Title

Quantitative analysis of molecular interactions in RNA trafficking in vivo and in silico

Date of Completion

January 2008

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Comprehensive understanding of any cell biological system requires: identification of all molecular components in the system, determination of all molecular interactions among components of the system, measurement of concentrations and diffusion coefficients for all components of the system and binding constants for all molecular interactions in the system, analysis of system behavior after perturbation of specific molecular components or specific molecular interactions and quantitative modeling of system behavior (Pollard, 2003). In this thesis we will describe new methods to complete this agenda and application of these methods to quantitative analysis of RNA trafficking in live cells. ^ The first chapter provides a general introduction to analysis of cell biological systems and RNA trafficking. ^ The second chapter describes a new binucleate cell assay for quantitative analysis of transformations that RNA undergoes during RNA trafficking. A Virtual Cell model for RNA trafficking using quantitative parameters measured in the binucleate cell assay is used to estimate parameters of RNA trafficking that cannot be measured experimentally. ^ The third chapter describes a general method using fluorescence correlation spectroscopy (FCS), western blotting and GFP fusions to measure concentrations and molecular interactions of proteins in live cells and application of the method to analyze splicing factors (Magoh and Y14), cap binding protein (CBP20) and granule scaffold protein (TOG). ^ The fourth chapter describes modifications to make the methods described in chapters 2 and 3 more generally applicable. ^