Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Thomas Blumenthal

Second Advisor

Chris Link

Third Advisor

Dick Davis

Fourth Advisor

Tin Tin Su

Fifth Advisor

Kevin Jones

Abstract

Tar-DNA Binding Protein 43 (TDP-43) was first discovered as a negative regulator of HIV transcription. Subsequently, TDP-43 was found as the major protein component of ubiquitin positive aggregates in a variety of neurodegenerative diseases, including ALS. Intensive study of TDP-43 function indicates that this protein plays a direct or indirect role in a large number of RNA-mediated processes. However, the basic function of TDP-43 and its connection to neurodegeneration remains elusive. In this thesis, I have undertaken the goal of discovering the basic biological function of TDP-43. I show that one of the basic functions of TDP-43 is to limit the accumulation of double stranded RNA (dsRNA). TDP-43 is required to maintain the abundance of transcripts with potential dsRNA structure and in the absence of TDP-43 nuclear dsRNA accumulates. I show that the dsRNA structure/stability of multiple transcript types is controlled by TDP-43 and that TDP-43 associates with these transcripts cotranscriptionally.

Using this knowledge, I then go on to characterize the molecular and phenotypic consequences of dsRNA accumulation in TDP-43 knockdown/deletion. I find that TDP-43 deletion in worms results in a severe chemotaxis defect, which is due to processes that metabolize dsRNA. Utilizing published data sets of TDP-43 knockdown in mice, I show that deletion of this protein in mammals results in the up-regulation of type I interferon response genes, which is a major consequence of increased dsRNA in mammals.

I also show that TDP-43 deletion in worms results in an increased level of A-to-I RNA editing, likely as a result of increased dsRNA structure/stability. Preliminary data indicates that mammalian TDP-43 also controls A-to-I RNA editing, but that this could be due in part to TDP-43 controlling the processing of the ADAR transcripts. Finally, I show evidence suggesting that TDP-43 may associate with inosine-containing RNA.

I also discover that TDP-43 deletion worms are differentially sensitive to RNA interference and that this sensitivity is due to a heightened response to nuclear RNAi or Transcriptional Gene Silencing (TGS). I show that worm TDP-43 associates in the TGS complex in an RNA-dependent manner. In the absence of worm TDP-43, the abundance of small interfering RNAs (siRNA) increases, but these siRNA are likely nonfunctional as the abundance of the transcripts they target is also increased in TDP-43 mutants. Importantly, I show that many repeat regions of the genome that fail to undergo TGS in TDP-43 mutants have expanded the DNA.

Finally, I show that worm TDP-43 maintains snoRNA/rRNA processing. In the absence of TDP-43 function, an extended form of U3 snoRNA as well as rRNA processing intermediates/defects accumulate. I provide suggestive evidence that TDP-43 may control rRNA processing by association with the nuclear exosome component, RRP6 and/or by limiting the stability of snoRNA/rRNA interactions. Consistent with the observed processing defect in rRNA, I show that TDP-43 deletion animals are sensitive to 5-florouracil (5-FU), a chemical that confers differential sensitivity in nuclear exosome mutants. As rRNA is a major component of ribosomes, I also investigated the effect of worm TDP-43 deletion and associated rRNA processing defects on protein aggregation, a hallmark of ALS. I find that both 5-FU treatment and worm TDP-43 deletion result in increased aggregation of a poly(Q) containing protein, potentially linking defects in rRNA processing with defective ribosomes/translation.

Saldi_Thesis_ChapterII_Tables.xlsx (2223 kB)
Chapter II Tables

Saldi_Thesis_ChapterIII_Tables.xlsx (177 kB)
Chapter III Tables

Saldi_Thesis_ChapterIV_Tables.xlsx (431 kB)
Chapter IV Tables

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