Identification of a cis-acting element involved in the regulation of BACE1 mRNA alternative splicing.

beta-site APP cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step during proteolysis of the beta-amyloid precursor protein, a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, and cis-acting elements important for its regulation have not been identified. We constructed and compared several BACE1 minigenes and found that BACE1 sequence from exon 3 through exon 5 was required for minigenes to undergo correct splicing. Minigene splicing was validated by showing specific splicing inhibition upon splice site mutation. Furthermore, we showed that mutation of the minigene at a predicted exonic splicing enhancer in exon 4 of BACE1 increased exon 4 skipping. Therefore, we have for the first time found evidence of a regulatory site involved in BACE1 alternative splicing, and these data indicate that minor sequence changes can dramatically alter BACE1 alternative splicing.

Promotion of BACE1 mRNA alternative splicing reduces amyloid beta-peptide production.

Production of the amyloid beta-peptide (Abeta) via sequential proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretases is strongly implicated in the pathogenesis of Alzheimer disease. The beta-secretase that executes the first cleavage event is a transmembrane aspartyl protease known as beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). BACE1 pre-mRNA is alternatively spliced through the use of alternative splice sites in exons 3 and 4, although the significance of these splicing events is unclear. Here, we quantitatively measured relative levels of BACE1 transcripts and identified a novel splice variant of BACE1. We found a subtle but significant difference in BACE1 splicing between brain and pancreas, indicating the cellular environment can affect BACE1 alternative splicing. Furthermore, we have shown that BACE1 proteins translated from alternatively spliced transcripts have dramatically reduced beta-secretase activity and promotion of BACE1 alternative splicing reduces Abeta production. These findings illustrate the importance of BACE1 alternative splicing in affecting the level of Abeta produced in cells and suggest that targeting regulation of BACE1 alternative splicing is a potential therapeutic strategy for lowering beta-secretase activity.

DNA replication of mitotic chromatin in Xenopus egg extracts.

Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a completely soluble replication system derived from Xenopus egg extracts to show that Cdk1/cyclin B also can support DNA replication. The ability of mitotic Cdks to drive DNA replication raises the question of whether DNA replication is possible in mitosis. To address this question, chromatin containing prereplication complexes was driven into mitosis with Cdk1/cyclin B. Strikingly, upon addition of a replication extract, the chromatin underwent a complete round of DNA replication. Replicating mitotic chromosomes became visibly decondensed, and, after DNA replication was complete, they recondensed. Our results indicate that there is extensive overlap in the substrate specificity of the major metazoan Cdk/cyclin complexes and that mitosis is not fundamentally incompatible with DNA replication. The results suggest that origins that fail to initiate DNA replication in S phase might still be able to do so in mitosis.