Doctor of Philosophy, The Ohio State University, 2007, Molecular, Cellular, and Developmental Biology

The genetic information transfer pathway, which includes transcription, precursor messenger RNA (pre-mRNA) processing, mRNA export, translation, and mRNA turnover, is a highly integrated process in higher eukaryotes. This integration is mainly achieved by the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (Pol II), which serves as a platform for recruiting RNA processing factors. In yeast Saccharomyces cerevisiae, however, support for the coupling of transcription and splicing is far less assuring. Here we show, via our studies of Sub2p, a DExD/H-box protein essential for both splicing and mRNA export, that transcription and splicing are functionally coupled in yeast. The DExD/H-box proteins are often referred to as RNA helicases, but are increasingly viewed as ribonucleoprotein ATPases (RNPases) that can remodel specific RNPs in vivo. We first show that specific alterations of the intron branch-site binding protein (BBP) can eliminate the requirement for Sub2p, thus illuminating a principal role for Sub2p to remove BBP and Mud2p from the branch-site region. Unexpectedly, we uncovered a panoply of genetic and chemical perturbations of the yeast transcription machinery that can also bypass Sub2p's requirement. Chromatin-immunoprecipitation (ChIP) analysis revealed that these perturbations significantly reduce the effectiveness of co-transcriptional recruitment of BBP, thereby alleviating the lethal outcome of losing Sub2p. These findings also suggest a potential selective advantage for yeast to exploit modifications in a kinetically coupled upstream pathway to offset the catastrophic loss of key components in the downstream pathways.

Committee: Tien-Hsien Chang (Advisor); Venkat Gopalan (Other); Paul Herman (Other); Amanda Simcox (Other) Subjects: Biology, Molecular

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

DEAD-box proteins are involved in virtually all aspects of eukaryotic RNA metabolism. As members of the helicase superfamily 2 (SF2), DEAD-box proteins utilize ATP hydrolysis to unwind RNA, assemble large protein complexes on RNA, and remodel RNA protein complexes (RNPs). In the cell however, DEAD-box proteins function in the context of RNPs during processes such as splicing and translation. How the biochemical activities of DEAD-box proteins are utilized in a physiological setting is an important and central question in RNA metabolism. In this thesis, we address this issue by examining RNP remodeling and cofactor modulation by the DEAD-box protein Ded1p.We demonstrated that Ded1p did not actively displace the RNA binding proteins U1A and TRAP from their cognate RNA binding sites. Additionally, we established that the context of a RNP determined active displacement by Ded1p and propose a model for RNP remodeling by DEAD-box proteins. We found that that an inability to actively displace other proteins from RNA can provide non-sequence specific DEAD-box proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. We further identified a physiologically relevant interaction between Ded1p and the translation initiation factor eIF4G. We showed that eIF4G did not modulate Ded1p ATPase activity, but did inhibit strand separation by Ded1p. Interestingly, Ded1p greatly increased eIF4G's affinity for RNA even to RNAs too small for eIF4G to bind alone. Our results suggest that Ded1p's biochemical activities facilitate eIF4G RNA binding. We propose a basic model for Ded1p enhancement of eIF4G RNA binding which may be relevant for Ded1p's role during translation initiation.

Committee: Eckhard Jankowsky (Advisor); Martin Snider (Committee Chair); Hung-Ying Kao (Committee Member); Pieter deHaseth (Committee Member) Subjects: Biochemistry; Molecular Biology