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Investigation of free U6 small nuclear ribonucleoprotein structure and function Dunn, Elizabeth Arlene


Eukaryotes differ from other domains of life in many respects, but at the level of gene architecture, it is the presence of interrupting sequences in their genes that serve as the defining feature. These must be removed with absolute precision in order to maintain the reading frame during translation of the encoded protein; thus it is not surprising that errors in this process, known as precursor messenger RNA (pre-mRNA) splicing, have been linked to a wide variety of human diseases. U6 small nuclear ribonucleoprotein (snRNP) is an essential component of the spliceosome, the large RNA-protein complex that is responsible for catalyzing pre-mRNA splicing. Although U6 small nuclear RNA (snRNA) plays a critical role in catalyzing the splicing reactions, very little is known about the mechanism of converting catalytically inert U6 snRNA in free U6 snRNP into a catalytic component of the assembled spliceosome. Here I present a model for free U6 snRNA secondary structure in free U6 snRNP that suggests that U6 becomes active for splicing through a mechanism that is dependent on its interaction with a second splicing factor, U4 snRNA. I propose that the U6 snRNP-associated protein, Prp24, is responsible for retrieving U6 snRNA from the disassembling spliceosome following splicing of a substrate, and then holds U6 snRNA in a conformation that masks catalytic sequences. I provide evidence, both from the literature and from my own genetic analysis, that the first two RNA Recognition Motifs of Prp24 bind a region of U6 snRNA known as the telestem, presenting U6 in a manner that is favorable for interaction with U4 snRNA. As a step toward solving the crystal structure to test this model, I have developed a system for the simultaneous recombinant expression of all components of U6 snRNP from a single expression vector, followed by purification of the pre-formed complex under non-denaturing conditions. I have subjected these particles to low-resolution negative stain electron microscopy and have also obtained a small angle X-ray scattering model of a sub-complex of free U6 snRNP, the LSm complex. This work has laid the foundation for understanding the structure/function relationship for U6 snRNP.

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