Small Interfering RNA-mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii

Authors

• Xinrong Ma, University of Nebraska-LincolnFollow

First Advisor

Heriberto Cerutti

Date of this Version

5-2013

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy

Major: Biological Sciences

Under the supervision of Professor Heriberto Cerutti

Lincoln, Nebraska, May 2013

Comments

Copyright 2013, Xinrong Ma. Used by permission

Abstract

RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses. RNAi can function by transcriptional silencing, mRNA target cleavage, translation repression and/or DNA elimination. In this study, we used the unicellular green alga Chlamydomonas reinhardtii as a model system to study RNAi-mediated translation repression. We demonstrated that small RNAs (sRNAs) generated from exogenously introduced inverted repeat transgenes, with perfect complementarity to the 3’UTR of a target transcript, can inhibit protein synthesis, without or with only minimal mRNA destabilization. In addition, there are no changes in the polyadenylation status of sRNA-repressed transcripts. Moreover, the translationally repressed mRNAs remain associated with polyribosomes, suggesting that sRNA-mediated silencing occurs at a post-initiation step of translation. Intriguingly, we consistently observed reduced sensitivity of the ribosomes associated with these repressed transcripts to inhibition by antibiotics such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in Chlamydomonas may involve alterations to the function/structural conformation of translating ribosomes. Additionally, since sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, this mechanism may have been a feature of an ancestral RNAi machinery.

Advisor: Heriberto Cerutti