Biochemical Studies Of Messenger Ribonucleic Acid Degradation Mechanisms

Abstract

The Carbon Catabolite Repressor Protein 4-Negative on TATA-less, or CCR4-NOT, is a multisubunit cytoplasmic complex, which is conserved in all eukaryotes. CCR4-NOT has multiple known functions in a cell but its principal molecular role is the enzymatic shortening of the polyadenosine, or poly(A), tail at the 3′ termini of messenger ribonucleic acids (mRNAs). This process is called deadenylation and it results in mRNA degradation and translational repression. The human CCR4-NOT complex comprises two catalytic and six non-enzymatic subunits organized into four functional modules. These modules and their subunits are highly functionally specialized. A major unresolved question is whether non-enzymatic modules and their subunits are directly involved in the deadenylation process. To address this, I developed a procedure for the biochemical reconstitution of the entire human CCR4-NOT complex from purified recombinant proteins. The reconstitution protocol ensured biochemical tractability of the complex for experimentation and laid the foundation for systematical dissection of contributions of the individual modules and subunits of the CCR4-NOT complex to the deadenylation process. My development of the reconstitution of the human CCR4-NOT complex also facilitated several collaborative studies within my group and with other laboratories. These studies address mechanistic questions of the kinetics of deadenylation in different sequence contexts, targeted deadenylation by direct recruitment of CCR4-NOT by RNA-binding specificity factors, and how CCR4-NOT interacts with other factors involved in the 5′-3′ mRNA decay pathway. Collectively, the results of my doctoral thesis work are a contribution to the field of RNA biology through a detailed biochemical assessment of the molecular mechanisms that regulate the culmination of the gene expression pathway.

Dissertation ist gesperrt bis 12.12.2024 !