The microRNA 15/16 cluster downregulates protein repair isoaspartyl methyltransferase in hepatoma cells: implications for apoptosis regulation.

Asparaginyl deamidation, a spontaneous protein post-biosynthetic modification, determines isoaspartyl formation and structural-functional impairment. The i s o a s p a r t y l p ro t e i n c a r b o x y l - O -methyltransferase (PCMT; EC 2.1.1.77) catalyzes the repair of the isopeptide bonds at isoaspartyl sites, preventing deamidationrelated functional impairment. Protein deamidation affects key apoptosis mediators, such as BclxL, thus increasing susceptibility to apoptosis, while PCMT activity may effectively counteract such alterations. Aims of this work was to establish the role of RNAi as a potential mechanism for regulating PCMT expression and its possible implications in apoptosis. We investigated the regulatory properties of miR15a/16-1 cluster on PCMT expression on HepG2 cells. MiR15a or 16-1 transfection, as well their relevant antagonists, showed that PCMT is effectively regulated by this miRNA cluster. The direct interaction of these two miRNAs with the seed sequence at the 3 UTR of PCMT transcripts was demonstrated by luciferase assay system. The role of PCMT downre g u l a t i o n i n conditiong t h e susceptibility to apoptosis was investigated by using various specific snRNA approaches, in order to prevent non PCMT specific pleiotropic effects to take place. We found that PCMT silencing is associated with an increase of the BclxL isoform which has been reported to be inactivated by deamidation, thus making cells more susceptible to apoptosis induced by cisplatinum. We conclude that PCMT is effectively regulated by the miR15a-16-1 cluster and is involved in apoptosis by preserving the structural stability and biological function of BclxL from deamidation. Control of PCMT expression by snRNAs may thus represent a late check point in apoptosis regulation.