Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular Biology & Biotechnology

Date of Defense

5-8-2008

Graduate Advisor

Marc Spingola, PhD

Committee

Marc Spingola, Ph.D.

Wendy Olivas, Ph.D.

Teresa Thiel, Ph.D.

Wesley Harris, Ph.D.

Abstract

The transition from mitosis to meiosis in the yeast Saccharomyces cerevisiae requires a significant change to gene expression profiles. Regulation of pre-messenger RNA splicing patterns during meiosis assists in this transition by fine tuning expression of essential meiotic genes. Produced only during meiosis, Mer1p is linked to the splicing of at least three mRNAs: MER2, MER3, and AMA1. Previous evidence suggests that Mer1p activates splicing by directly recruiting snRNPs or stabilizing intermediate splicing complexes formed on pre-mRNA that contains an intronic Mer1p enhancer element. However, some splicing factors, especially accessory/non-snRNP factors, have critical roles in retaining unspliced pre-mRNAs in the nucleus. I tested if Mer1p may indirectly regulate splicing by preventing the export of pre-mRNAs to the cytoplasm and also demonstrated that a second subunit of the Retention and Splicing (RES) complex, Bud13p, has transcript-specific effects on Mer1p-activated splicing. The results indicated that Mer1p can retain unspliced pre-mRNA in the nucleus; however, nuclear retention could not be uncoupled from splicing activation. In the absence of Mer1p, the AMA1 pre-mRNA is exported to the cytoplasm, translated, but not subjected to nonsense-mediated decay (NMD) despite a premature stop codon in the intron. A novel role for the Mer1p activation domain was revealed by a two-hybrid interaction with Prp39p, an essential U1 snRNP protein. This suggests the initial contact between Mer1p and the spliceosome occurs during commitment complex assembly. Collectively, these data imply that Mer1p can retain pre-mRNAs in the nucleus only by facilitating their interaction with the spliceosome and support models for cytoplasmic degradation of unspliced pre-mRNAs that fail to assemble into spliceosomes in yeast. A two-hybrid analysis of U1 snRNP proteins and other early splicing factors tested 460 possible interactions and the several novel interactions reported here indicate a revised model for U1snRNP structure.

OCLC Number

547456865

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