DECIPHERING THE ROLE OF TDP-43 IN TRANSLATION

picture of Antony Pearson presenting his/her poster: DECIPHERING THE ROLE OF TDP-43 IN TRANSLATION

Antony Pearson , Daniela C. Zarnescu

DECIPHERING THE ROLE OF TDP-43 IN TRANSLATION

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. It involves the death of motor neurons, leading to paralysis, respiratory failure, and death, usually within 2-5 years of onset of clinical symptoms. Recent studies implicate alterations in RNA metabolism as key events in the pathogenesis of ALS. An RNA-binding protein known as TAR DNA Binding Protein, TDP-43, has been linked to ALS. Several mutations in TDP-43 have been identified as a possible primary cause of neurodegeneration in ALS. We hypothesize that TDP-43 mutations result in neurodegeneration by affecting the translation of specific mRNA targets. 

We examined 4 mutant human TDP-43 variants known to cause ALS in humans (D169G, G298S, A315T, and N345K) as well as wild-type human TDP-43. Using a Drosophila model, we expressed each variant in all motor neurons using the tissue-specific GAL4-UAS system under the control of the D42 GAL4 driver. Flies of each genotype were used in polysome fractionation experiments. Using a flow-through spectrometer, ribonucleoproteins, ribosomal subunits, monosomes, and polysomes were separated and identified with high resolution. We observed some differences between profiles for different genotypes, indicating changes in global translation. 

Future work includes coupling the polysome fractionation to RNA sequencing and Western blotting to gain insight into how TDP-43 affects protein-protein interactions and translation of specific mRNAs. Separating RNAs that fall in the ribonucleoprotein region from actively translated RNAs that are associated with polysomes and then sequencing them separately will tell us the effect of each TDP-43 mutation in how it affects the translation of its target RNAs. Coupling polysome fractionation to Western blotting will allow us to see how mutations in TDP-43 affect the distribution of TDP-43 and its putative binding partners in the polysome profile.

This research was supported in part by a grant to the University of Arizona from the Beckman Foundation.

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