Citation
- Authors: Kattan FG. et al.
- Year: 2023
- Journal: Open Biol. 13 230158
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: HEK-293A
- Name: SK-N-SH
Description: Human bone marrow neuroblastoma
Method
Human SK-N-SH and HEK293A cells were grown in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified 5% CO2 incubator and transfected at plating with plasmids using the jetOPTIMUS according to Polyplus recommendation. EmGFP plasmid transfection indicated that efficiency was approximately 80% for both cell lines at 48 h. Cells were harvested 48 h after transfection.
Abstract
Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.