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Defining the Properties and Toxicity Mechanisms of Non-Canonical Translation Products Associated with ALS and Huntington’s Disease Using C. elegans

Rudich, Paige (2019) Defining the Properties and Toxicity Mechanisms of Non-Canonical Translation Products Associated with ALS and Huntington’s Disease Using C. elegans. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Expanded guanine/cytosine (G/C)-rich nucleotide repeats are the underlying
genetic cause of many age-dependent neurodegenerative diseases. An emerging
mechanism underlying disease pathology is an unusual type of protein translation called
Repeat-Associated non-AUG (RAN) translation. RAN translation requires extended G/Crich
repeats and occurs independently of a canonical start codon, allowing translation in
all three reading frames. Antisense RNA from G/C-rich repeats also gives rise to RAN
products, causing up to six distinct protein products from one repeat expansion. The
toxicity of RAN products in vivo is beginning to be explored. We created codon-varied
RAN product models for two different repeats: an intronic GGGGCC repeat expansion
that is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS), and the
CAG repeat expansion that causes Huntington’s disease (HD).
In our C. elegans ALS model, we discovered that the arginine-containing
dipeptides, proline-arginine (PR) and glycine-arginine (GR), were the most toxic
dipeptides. PR and GR exhibited age-dependent toxicity when expressed in multiple cell
types, including motor neurons. Both PR and GR exhibited nuclear localization that was
necessary for toxicity. An unbiased whole-genome RNAi screen for suppressors of PR
toxicity identified twelve genes. Four of the genes were previously identified in PR
modifier screens performed in other systems, suggesting mechanisms of PR toxicity are
My studies are the first to model codon-varied CAG RAN polypeptides in a
multicellular animal. Every polypeptide, except for polyLeucine (polyLeu), formed
immobile protein aggregates at ~38 repeats. Surprisingly, polyLeu was the most toxic
HD RAN polypeptide in every tissue studied. A forward mutagenesis screen combined
with a candidate RNAi screen identified three genes that suppressed polyLeu toxicity.
Two of the genes encoded transmembrane proteins, and the third gene encoded a
deubiquitinase enzyme, suggesting that polyLeu toxicity occurs via disrupted folding of
transmembrane proteins. Understanding how different RAN products contribute to HD
and ALS is vital for developing appropriate treatments for these diseases.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Rudich, Paigepaige.d.rudich@gmail.compad680000-0001-6979-9701
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBrodsky, Jeffreyjbrodsky@pitt.edujbrodsky
Committee MemberPalladino, Michaelmjp44@pitt.edumjp44
Committee MemberPandey, Udaiudai@pitt.eduudai
Committee MemberTsang, Michaeltsang@pitt.edutsang
Committee MemberYanowitz, Judithjly23@pitt.edujly23
Thesis AdvisorLamitina, Toddstl52@pitt.edustl52
Date: 4 September 2019
Date Type: Publication
Defense Date: 15 May 2019
Approval Date: 4 September 2019
Submission Date: 18 July 2019
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 213
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Amyotrophic Lateral Sclerosis, C. elegans, Huntington's disease, PolyQ, RAN Translation,
Date Deposited: 04 Sep 2019 13:50
Last Modified: 04 Sep 2019 13:50


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