Overexpression of high-mobility group A protein 1a (HMGA1a) causes aberrant exon 5 skipping of the Presenilin-2 (PS2) pre-mRNA, which is almost exclusively detected in patients with sporadic Alzheimer's disease. An electrophoretic mobility shift assay confirmed aberrant U1 small nuclear ribonucleoprotein particle (snRNP)-HMGA1a complex formation (via the U1-70K component), with RNA containing a specific HMGA1a-binding site and an adjacent 5' splice site. Psoralen cross-linking analysis demonstrated that the binding of HMGA1a adjacent to the 5' splice site induces unusually extended association of U1 snRNP to the 5' splice site. As a result, spliceosome assembly across either the intron or the exon is arrested at an early ATP-independent stage. We conclude that the HMGA1a-induced aberrant exon skipping is caused by impaired dissociation of U1 snRNP from the 5' splice site, leading to a defect in exon definition. The proposed molecular mechanism has profound implications for other known posttranscriptional modulation strategies in various organisms, all of which are triggered by aberrant U1 snRNP binding.