Investigating the roles of phosphorylation in modulating Argonaute 2 activity and localization

• Author / Creator

  Lopez Orozco, Joaquin

• RNA interference (RNAi) has enabled the study of gene function with unprecedented specificity and reliability. Almost twenty years after the discovery of RNAi, we now know that this process is carried out by a set of evolutionarily conserved proteins. In mammals, these proteins form part of the miRNA pathway that controls the expression of most protein-encoding genes. At the core of the miRNA pathway are Argonaute proteins, which are guided by miRNAs to downregulate the expression of more than 50% of all mRNAs. While there is a vast literature describing how miRNAs regulate numerous cellular pathways, comparatively less is known about how miRNA pathways are modulated. Because of their role in gene expression on a global level, miRNA pathways must clearly be subjected to extensive regulation. Recent findings indicate that post-transcriptional modifications of Argonaute proteins are important for this process. Human Argonaute 2 is phosphorylated on at least seven amino acid residues but we know very little about the function consequences of these modifications. Furthermore, very little is known about the kinases and phosphatases that catalyze these modifications. One unexpected finding in my thesis research is that changes in phosphorylation can lead to profound changes in the localization of Argonaute proteins without dramatically affecting their gene-silencing functions. Using a screen biased to detect kinases that regulate the late stages of RNAi, I identified a large number of kinases that decrease or increased RNAi activity. Few kinases were found to inhibit RNAi activity, but interestingly, the majority were tyrosine kinases. Among this group, FGFR3 was further examined and its effects on RNAi activity were studied in detail. Of note, I showed that stimulation of multiple FGFR family members inhibit RNAi, likely through phosphorylation of Argonaute proteins. Finally, I showed that Argonaute 2 interacts with the serine/threonine phosphatase PP1 and that elevated expression of PP1 inhibits RNAi activity.

• Subjects / Keywords

  • FGFR
  • RNAi
  • Phosphorylation
  • Argonaute 2

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3HQ3SF3P

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.