title: Genetic diversity at the UGT1 locus is amplified by a novel 3' alternative splicing mechanism leading to nine additional UGT1A proteins that act as regulators of glucuronidation activity.
abstract: BACKGROUND
  The gene UGT1 encodes phase II detoxification proteins involved in the elimination of small hydrophobic substances of both endogenous and exogenous origin. To date, nine functional UGT1A proteins are known to be produced from a single gene composed of alternative first exons shared with four common exons. Recently, a novel exon (referred to as exon 5b) was identified in the common shared region.
  
  
  RESULTS
  We now reveal a novel alternative splicing mechanism and demonstrate that the exon 5a and the new exon 5b are alternatively spliced, generating several variant mRNAs and up to nine previously unknown variant UGT1A proteins, referred to as isoforms 2 or i2. Isoform-specific RT-PCR analyses reveal that the alternatively spliced mRNAs are widely distributed in human tissues. Immunoreactive proteins at the predicted molecular weight of approximately 45 kDa were confirmed in microsomes of human tissues using antibodies against UGT1A1 and anti-UGT1A7/8/9/10. Functional enzyme assays demonstrate that i2 proteins containing exon 5b are enzymatically inactive. On the other hand, co-expression experiments of i2 of UGT1A1, UGT1A7, UGT1A8 and UGT1A9 with their classical isoform 1 homologs results in a significant repression (15 to 79%) of UGT1A_i1-mediated drug metabolism.
  
  
  CONCLUSIONS
  The UGT1A isoforms 2 act as negative modulators of their isoform 1 homologs in microsome preparations, revealing a new regulatory mechanism of the glucuronidation pathway. Findings further provide the first direct evidence of a novel alternative splicing mechanism at the 3' end of the UGT1 locus that further increases the number of proteins derived from this single gene.
authors.affiliation: Laboratory of Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Faculty of Pharmacy, Laval University, Québec, Canada.
authors.forename: Hugo
authors.initials: H
authors.lastname: Girard
authors.forename: Eric
authors.initials: E
authors.lastname: Lévesque
authors.forename: Judith
authors.initials: J
authors.lastname: Bellemare
authors.forename: Kim
authors.initials: K
authors.lastname: Journault
authors.forename: Bertrand
authors.initials: B
authors.lastname: Caillier
authors.forename: Chantal
authors.initials: C
authors.lastname: Guillemette
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D014644:Genetic Variation; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008252:Macaca fascicularis; D008957:Models, Genetic; D010597:Pharmacogenetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D000099148:UGT1A1 Enzyme
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D020719:Glucuronides; D014453:Glucuronosyltransferase; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme