Date of Graduation
5-2016
Document Type
Dissertation (PhD)
Program Affiliation
Genes and Development
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Dr. Leslie Krushel
Committee Member
Dr. Gilbert Cote
Committee Member
Dr. William Mattox
Committee Member
Dr. Richard Lloyd
Committee Member
Dr. Sadhan Majumder
Committee Member
Dr. Pierre McCrea
Abstract
The translation of most eukaryotic mRNAs occurs in a cap-dependent manner. However, a subset of mRNAs are capable of initiating translation in a cap-independent manner by utilizing sequences in their 5’ UTR called IRES. It was previously shown that the 5’ UTR of the tau mRNA contains an IRES. In this study I show that IRES dependent translation of tau IRES is regulated at multiple levels in order to regulate the expression of the tau protein.
Tau protein is ubiquitously expressed but is concentrated in the brain. In this study, I utilized neural and non-neural cell lines to show that tau IRES is utilized differently (in some cases, up to 50% of total tau translation) depending on the cell type. For many IRES containing mRNAs, IRES activity is enhanced in conditions when cap-dependent translation is shut down, such as during cellular stress and mitosis. In this study, I show that tau IRES activity is upregulated during increased iron, poly (I:C), and extracellular Aβ exposure, which are stress conditions commonly observed in neurodegenerative diseases. Further, I show that tau IRES is differentially regulated by various upstream stimuli through their downstream signaling kinases. However, a comparison of the effect of various signaling pathways on tau and APP IRES suggested that the specific regulation of these IRESes occur downstream of mTOR signaling.
Most IRESes require binding by certain non-canonical factors called IRES trans acting factors (ITAFs) for internal initiation. ITAFs can be positive or negative, thus enhancing or inhibiting IRES function. Examination of sequences in the tau 5’ UTR led us to analyze four different RNA binding proteins as putative ITAFs for tau. Out of these, I identified two proteins – polypyrimidine tract binding protein (PTB) and neural PTB (nPTB) as inhibitory ITAFs of tau IRES. Altering the expression of PTB and nPTB in vitro and in cells negatively influenced tau IRES activity and protein expression.
Along with sequences in the 5’ UTR, the sequences in the 3’ UTR of an mRNA may also affect its translation, either through direct interaction between the RNA sequences, or through interaction by RNA binding proteins. In this study, I show that the tau 3’ UTR enhances IRES-dependent translation of tau, and this interaction requires the entire tau 3’UTR. Overall, I show that the tau IRES is a unique tool utilized by the mRNA to regulate tau protein expression.
Keywords
Translation, IRES, tau, Alzheimer's Disease, Polypyrimidine tract binding protein, 5' UTR