a-Synuclein plays a major role in familial and sporadic Parkinson's disease. Unraveling the mechanisms that promote a-synuclein aggregation is essential to understand the formation of Lewy bodies and possibly the mechanisms involved in dopaminergic cell death. a-Synuclein is known to be ubiquitylated in Lewy bodies, but the role of a-synuclein ubiquitylation has been mysterious. We now report that the E3 ubiquitin-ligase SIAH directly interacts with and monoubiquitylates a-synuclein, and promotes its aggregation both in vitro and in vivo. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates a-synuclein at lysines 12, 21 and 23, which were previously shown to be ubiquitylated in Lewy bodies. Additionally, SIAH ubiquitylates lysines 10, 34, 43 and 96 as well. Suppression of SIAH expression by shRNA to SIAH-1 and SIAH-2 abolished a-synuclein monoubiquitylation in human dopaminergic cells, indicating that endogenous SIAH ubiquitylates a-synuclein. Moreover, SIAH co-immunoprecipitated with a-synuclein from brain extracts, indicating a physiological interaction. Inhibition of proteasomal, lysosomal and autophagic pathways, as well as overexpression of an ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of a-synuclein by SIAH, suggesting that different proteolytic pathways and deubiquitinases play a role in regulating a-synuclein monoubiquitylation. Additionally, monoubiquitylation increased the aggregation of a-synuclein in vitro as determined by Western blot and electron microscopy assays. At the electron microscopy level, monoubiquitylated a-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased a-synuclein aggregation in vivo as observed by the increased formation of a-synuclein inclusion bodies within dopaminergic cells. The formation of a-synuclein inclusions was prevented when endogenous SIAH expression was suppressed by shRNAs to SIAH-1 and SIAH-2. Furthermore, inclusions formed by monoubiquitylated a-synuclein were not protective to SH-SY5Y cells. Instead, they were toxic to some extent. Our data suggest that monoubiquitylation represents a possible trigger event for a-synuclein aggregation and Lewy body formation.