The UGT1A1 glucuronidation pathway is regulated by a new mechanism of alternative splicing in the UGT1A gene

Hugo Girard, Éric Lévesque, Kim Journault, Johanie Lépine, Chantal Guillemette
Canada Research Chair in Pharmacogenomics, Laboratory of Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Faculty of Pharmacy, Laval University, 2705, boulevard Laurier, Québec, Qc, G1V 4G2, Canada

The UDP-glucuronosyltransferase enzyme 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. Here we reported the identification of a new UGT1A1 isoform, referred as isoform 2 (UGT1A1_i2) generated by an alternative splicing event involving an additional exon found at the 3' end of the UGT1A locus. The UGT1A gene structure is rather unusual and is characterized by 13 different versions of exon 1, which maybe alternatively spliced to the common exons 2-5. The objective of this study was to assess the expression profile of UGT1A1_i1 and i2 in various human tissues and to determine the function of this newly identified protein. Tissue distribution of both UGT1A1 isoforms was studied via RT-PCR and western blotting. To explore the subcellular localization of UGT1A1_i2, a serie of immunofluorescence analyses were performed with specific antibodies. The function of UGT1A1_i2 expressed alone or coexpressed with UGT1A1_i1 was assessed by enzymatic assays with microsomes from stably transfected HK293 cells. We demonstrate that the isoform 2 is differentially expressed in liver, kidney, colon and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Futhermore, interindividual variation was observed for the expression of UGT1A1_i2 in human hepatic microsome samples. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data demonstate that UGT1A1_i2, which lacks transferase activity when stably expressed alone, acts as a negative modulator of UGT1A1_i1 decreasing its activity by up to 78%. Co-immunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that the repression may occur via direct protein-protein interactions. Data reveal a novel alternative splicing mechanism at the UGT1A locus that amplifies the structural diversity of human UGT proteins and describes the identification of an additional post-transcriptional regulatory mechanism of the glucuronidation pathway.