Regulation of the UGT1A1 bilirubin-conjugating pathway: role of a new splicing event at the UGT1A locus.
UDP-glucuronosyltransferase 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. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3' end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99-amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that 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. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions.


CONCLUSIONS
Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway.
Lévesque
Eric
E
Laboratory of Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Faculty of Pharmacy, Laval University, Québec, Canada.
Girard
Hugo
H
Journault
Kim
K
Lépine
Johanie
J
Guillemette
Chantal
C
D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003106:Colon / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D000099148:UGT1A1 Enzyme
D018076:DNA, Complementary; D014453:Glucuronosyltransferase; D007527:Isoenzymes; D012333:RNA, Messenger; D000099148:UGT1A1 Enzyme