UNDERSTANDING HOW DHH1 AND CCR4-NOT COMPLEX REGULATE mRNA METABOLISM IN YEAST

Open Access
Author:
Dutta, Arnob
Graduate Program:
Cell and Developmental Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 17, 2010
Committee Members:
  • Joseph C. Reese, Dissertation Advisor
  • Joseph C. Reese, Committee Chair
  • David Scott Gilmour, Committee Member
  • Craig Eugene Cameron, Committee Member
  • Yanming Wang, Committee Member
  • Philip C. Bevilacqua, Committee Member
Keywords:
  • transcription
  • mRNA
Abstract:
The life of mRNA begins with transcription in the nucleus and ends with destruction in the cytoplasm. Several proteins work in unison to regulate the various stages of mRNA metabolism. The Ccr4-Not complex plays roles from the “birth” to the “death” of mRNAs. Dhh1, an evolutionarily conserved member of the DEAD box family in yeast, associates with the Ccr4-Not complex, and regulates both transcription and mRNA decay. Additionally, in response to cellular stress, Dhh1 localizes to P-bodies where it affects the destruction of mRNAs. By interacting with the translation machinery Dhh1 and its orthologs also play roles in translational repression. Further it also plays important roles in G1/S DNA damage checkpoint recovery. Given the many functions of Dhh1 in both the synthesis and decay of RNAs, this study seeks to examine the structural requirements for all of Dhh1’s functions. Biochemical analysis of Dhh1 reveals that this protein binds RNA with high affinity but displays poor ATPase activity in vitro as compared to other DEAD box helicases. By studying the biochemical activities of Dhh1, key residues involved in ATP and RNA binding, and ATP hydrolysis have been identified. Next, this study helps dissect out how inactivation of these various biochemical activities affects the functioning of Dhh1, in vivo. In vivo analysis of mutant alleles that affect the biochemical activities has revealed the important roles of ATP binding and hydrolysis in most of the functions of Dhh1. Interestingly, the weak ATPase activity of Dhh1 is in part due to extensive inter-domain interactions, disruption of which greatly enhances ATP hydrolysis. We hypothesize that Dhh1 activity is stimulated by cellular factors, which impart a tight regulation on the functioning of this very abundant helicase. The highly conserved Ccr4-Not complex has been ascribed many functions, from transcription regulation to mRNA decay. Initial studies described a role of this complex in preinitiation complex formation, but a number of studies have clearly shown Ccr4-Not mediates deadenylation of mRNAs and protein ubiquitylation in the cytoplasm. It is still not clear how Ccr4-Not regulates gene expression, and which of these functions are controlled directly by this complex. This study demonstrates that Ccr4-Not complex directly regulates RNAP II-dependent transcription elongation. Using purified Ccr4-Not complex and yeast RNAP II in an in vitro transcription system, the role of the Ccr4-Not complex in regulating transcription elongation has been studied. Studies show that the complex binds directly to the elongating RNAP II complex, which is partially dependent on the emerging transcript, and it also makes contacts with the emerging transcript. Using transcription run on assays, Ccr4-Not is shown to stimulate the resumption of elongation from paused RNAP II elongation complexes. Further, Ccr4-Not works co-operatively with positive elongation factor TFIIS to stimulate elongation through transcription blocks. The in vitro results are substantiated by in vivo studies, showing that Ccr4-Not co-purifies with RNAP II and associates with the open reading frames of transcribing genes. Together these studies support a model by which the Ccr4-Not complex directly associates with transcribing RNAP II and via its interaction with the emerging transcript positively regulates transcription elongation.