RNA interference (RNAi) is a newly discovered gene control pathway that silences gene expression in response to double-stranded RNA. This sequence-specific downregulation occurs at the level of mRNA stability, and is directed by the double-stranded RNA trigger itself. RNAi can be separated into two distinct steps; during the initiation phase, Dicer, a ribonuclease III family member, cleaves the long double-stranded RNA trigger into 21-23 nucleotide small interfering RNAs (siRNAs). In the subsequent effector phase, these siRNAs are incorporated into the RNA-induced silencing complex (RISC), where they direct the site-specific cleavage of complementary messenger RNA. Much of our understanding of this silencing pathway stems from studies using Drosophila melanogaster; however, how closely the biochemistry of this model organism mirrors human RNAi is not well understood. Furthermore, numerous fundamental questions remain regarding this pathway, such as what role, if any, Dicer plays in the second stage of RNAi. In this dissertation, I further characterize Dicer's role in the effector phase of RNA interference. UV crosslinking of Dicer proteins in Drosophila embryo lysates indicates that these proteins remain directly associated with siRNA throughout the RISC assembly process, and are present in the functional RISC. Furthermore, these proteins are required for RNAi activity. I also attempt to characterize the role of human Dicer in RISC assembly. Using native gel electrophoresis, I have identified a Dicer-containing complex that requires ATP for its formation. Furthermore, UV crosslinking experiments in human cell lysates suggest that themechanism for siRNA-loading into RISC may differ between humans and Drosophila.

Last modified

• 06/05/2018

Creator

• Janice Lynn Pellino

DOI

• https://doi.org/10.21985/N29T2B

Subject

• Interdepartmental Biological Sciences Program

Keyword

• Chemistry
• Biology

Date created

• 2007-05-09

Resource type

• Dissertation

Rights statement

• In Copyright