Regulation of Parkinson's disease-associated genes by Pumilio proteins and microRNAs in SH-SY5Y neuronal cells.

ABSTRACT

Parkinson's disease is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to Parkinson's disease including LRRK2, SAT1, and SNCA, the latter of which encodes the protein alpha-synuclein that aggregates in Lewy bodies as a hallmark of the disease. Overexpression of even wild-type versions of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood. Pumilio proteins belong to the highly conserved PUF family of eukaryotic RNA-binding proteins that post-transcriptionally regulate gene expression through binding conserved motifs in the 3' untranslated region (UTR) of mRNA targets known as PUF Recognition Elements (PREs). The 3'UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human Pumilio homologs (Pumilio 1 and Pumilio 2) in a neurodegenerative model cell line, SH-SY5Y, resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the Pumilio proteins. Some studies have indicated a relationship between Pumilio and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 each contain several putative microRNA-binding sites within the 3'UTR, some of which reside near PREs. Small RNA-seq and microRNA qPCR assays were performed in both wild type and Pumilio KD SH-SY5Y cells to analyze global and differential microRNA expression. One thousand four hundred and four microRNAs were detected across wild type and Pumilio KD cells. Twenty-one microRNAs were differentially expressed between treatments, six of which were previously established to be altered in Parkinson's disease patient samples or research models. Expression of ten miRs predicted to target LRRK2 and SNCA was verified by RT-qPCR. Collectively, our results demonstrate that Pumilios and microRNAs play a multi-faceted role in regulating Parkinson's disease-associated genes.