Role of TDP-43 and hnRNP Proteins in the Regulation of Different RNA Targets

Cappelli, Sara (2019). Role of TDP-43 and hnRNP Proteins in the Regulation of Different RNA Targets. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000efd5

Abstract

Heteregeneous ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins (RBPs) implicated in several steps of RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and turnover. Therefore, alteration of their physiological levels may lead to many pathological disorders, such as neurodegeneration and cancer.

In this thesis, we focused the attention on three hnRNP proteins, namely DAZAP1, hnRNP Q and hnRNP R, that we previously identified in Drosophila melanogaster as strong modulators of TAR DNA-binding Protein 43 kDa (TDP-43) activity, an RBP involved in Amyotrophyc Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD).

First of all, we evaluated the effects of their depletion on TDP-43-controlled mRNAs within human neuroblastoma SH-SY5Y cells and in a cellular model of TDP-43 loss-of- function. We found that DAZAP1 and, to a lesser extent, hnRNP Q were the most consistent modifiers of TDP-43 activity. Therefore, to examine the connection between DAZAP1 and TDP-43, we initially characterized the interaction between these two proteins by immunoprecipitation analysis, demonstrating that DAZAP1 does not bind TDP-43 but can bind TDP-43 controlled mRNAs. Next, we decided to identify all the potentially targets regulated by TDP-43 and DAZAP1, by looking at the transcriptome status of cells silenced for these two hnRNPs and we found differently expressed genes associated with neurodegeneration (ELAVL3 NOVA2, CELF5) and inflammation (TNF, TNFRSF9, ICAM1).

Finaly, we extended our analysis to the characterization of hnRNP Q and hnRNPR, since the functional rescue of TDP-43 alterations was described only for hnRNP Q, but not for hnRNPR. We investigated the subcellular distribution and profiled differentially expressed genes analysis from RNA-seq after their knockdown. Interestingly, despite their high sequence similarity, these two proteins show different cellular distribution and affect different cellular pathways, tipically associated with neurodegeneration (PENK, NGR3, RAB26, JAG1) and inflammatory response (TNF, ICAM1, TNFRSF9, ICAM5). In conclusion, our work provides insights on the involvement of the hnRNP family in controlling neuronal and inflammatory pathways, and suggests that the differential expression of these proteins could play an essential role in modulating the onset as well as the progression of neurodegenerative disorders, in particular when related to the TDP-43 proteinopathies.