Author ORCID Identifier

https://orcid.org/0000-0001-5525-3724

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Stefan Stamm

Abstract

Under pathophysiological conditions, the microtubule protein tau (MAPT) forms neurofibrillary tangles that are the hallmark of sporadic Alzheimer’s disease as well as familial frontotemporal dementias linked to chromosome 17 (FTDP-17). In this work, I report that MAPT forms circular RNAs through backsplicing of exon 12 to either exon 10 or exon 7 (12→10; 12→7), and that these circular RNAs are translated into proteins.

Using stable cell lines overexpressing the circular tau RNAs 12→7 and 12→10, we have discovered that the tau circular RNA 12→7 is translated in a rolling circle, giving rise to multiple proteins. This circular RNA has an endogenous start codon in exon 9 and no in-frame stop codon. We purified the protein by immunoprecipitation using a 3X flag tag that is upstream of the start codon and that will only be translated in a circular RNA. The circular RNA 12→10 does not have a start codon and is translated when mutations that cause FTDP-17 create a start codon (K317M and V337M) or ADAR (Adenosine Deaminase acting on RNAs) enzymes are present. In addition, we show that the 12→10 wild-type circular RNA can be translated due to RNA editing by ADAR1 and ADAR2. ADAR enzymes edit RNA sequences changing adenosines to inosines and are referred to as A-to-I editing (I = inosine). Inosines can be read as a guanosine, therefore editing an AUA sequence to AUI could create a new start codon in the RNA sequence.

The circular RNAs’ translated region is similar to a seed sequence, known as K18, that promotes tau aggregation and neurodegeneration. We hypothesize that the tau proteins generated from the circular RNAs contribute to tau aggregation, which causes Alzheimer’s Disease. We have designed siRNAs, specific for circular RNAs, as possible therapeutics. We show that the siRNAs target the circular tau RNAs backsplice junction, reducing their expression and preventing translation.

In summary, our data indicate that tau makes so far unknown circular RNAs that are unexpectedly translated into proteins that cause tau aggregations, a hallmark of tauopathies (AD and FTDP-17). Almost all FTDP-17 mutations are in the pre-mRNA regions contributing to circular RNA formation, suggesting they act through circular RNAs. Finally, we identified oligonucleotides that abolish circular RNA formation, pointing to a novel therapeutic avenue for tauopathies.

I am thankful for the partial funding of this project by the College of Medicine Excellence in Graduate Research Fellowship and the Max Steckler Award.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.018

Funding Information

University of Kentucky College of Medicine Excellence in Graduate Research Fellowship 2018-2019

University of Kentucky Max Steckler Fellowship 2019-2020

National Institute of Health/National Institute of Aging Understanding the influence of FTDP-17 mutation on human tau circular RNA formation and function 1R21AG067438-01 2016-2020

National Institute of Health/National Institute of Aging Role of Tau circular RNAs in tauopathies R21AG064626-01 2019-2021

Department of Defense Determine the role of human-specific tau circular RNAs in tauopathies W81XWH190502 2019-2022

Available for download on Sunday, September 05, 2021

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