The microRNA 15a/16-1 cluster down-regulates protein repair isoaspartyl methyltransferase in hepatoma cells: implications for apoptosis regulation.
Asparaginyl deamidation, a spontaneous protein post-biosynthetic modification, determines isoaspartyl formation and structure-function impairment. The isoaspartyl protein carboxyl-O-methyltransferase (PCMT1; EC 2.1.1.77) catalyzes the repair of the isopeptide bonds at isoaspartyl sites, preventing deamidation-related functional impairment. Protein deamidation affects key apoptosis mediators, such as BclxL, thus increasing susceptibility to apoptosis, whereas PCMT1 activity may effectively counteract such alterations. The aim of this work was to establish the role of RNAi as a potential mechanism for regulating PCMT1 expression and its possible implications in apoptosis. We investigated the regulatory properties of the microRNA 15a/16-1 cluster on PCMT1 expression on HepG2 cells. MicroRNA 15a or microRNA 16-1 transfection, as well as their relevant antagonists, showed that PCMT1 is effectively regulated by this microRNA cluster. The direct interaction of these two microRNAs with the seed sequence at the 3' UTR of PCMT1 transcripts was demonstrated by the luciferase assay system. The role of PCMT1 down-regulation in conditioning the susceptibility to apoptosis was investigated using various specific siRNA or shRNA approaches, to prevent non-PCMT1-specific pleiotropic effects to take place. We found that PCMT1 silencing is associated with an increase of the BclxL isoform reported to be inactivated by deamidation, thus making cells more susceptible to apoptosis induced by cisplatinum. We conclude that PCMT1 is effectively regulated by the microRNA 15a/16-1 cluster and is involved in apoptosis by preserving the structural stability and biological function of BclxL from deamidation. Control of PCMT1 expression by microRNA 15a/16-1 may thus represent a late checkpoint in apoptosis regulation.
Department of Biochemistry and Biophysics, Second University of Naples, Naples 80138, Italy.
Irene
I
Sambri
Department of Biochemistry and Biophysics, Second University of Naples, Naples 80138, Italy.
Rosanna
R
Capasso
Ceinge, Advanced Biotechnologies and School of Life Science, "Federico II" University, Naples 80138, Italy.
Piero
P
Pucci
First Division of Nephrology, School of Medicine and Surgery, Second University of Naples, Naples 80138, Italy.
Alessandra F
AF
Perna
Department of Biochemistry and Biophysics, Second University of Naples, Naples 80138, Italy. Electronic address: diego.ingrosso@unina2.it.
Diego
D
Ingrosso
D020413:3' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D004260:DNA Repair; D015536:Down-Regulation; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D035683:MicroRNAs / Q000737:chemistry*; D008969:Molecular Sequence Data; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000096:biosynthesis / Q000378:metabolism*; D034622:RNA Interference; D012689:Sequence Homology, Nucleic Acid; D051020:bcl-X Protein / Q000378:metabolism
D020413:3' Untranslated Regions; C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D051020:bcl-X Protein; C435725:PCMT1 protein, human; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase