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Molecular and Cellular Biology, September 2005, p. 7505-7521, Vol. 25, No. 17
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.17.7505-7521.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mammalian Peptidylglycine -Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

Fabienne Brenet,¹ Nadège Dussault,¹ Jonas Borch,² Géraldine Ferracci,³ Christine Delfino,¹ Peter Roepstorff,² Raymond Miquelis,^3, and L'Houcine Ouafik^1,*

Université de la Méditerranée, Aix-Marseille II, Laboratoire de Cancérologie Expérimentale, Inserm EMI 0359, Faculté de Médecine Secteur Nord, IFR Jean Roche, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France,¹ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark,² Université de la Méditerranée, Aix-Marseille II, FRE 2738 CNRS and Unité de Méthodologie des Interactions Moléculaire, Faculté de Médecine Secteur Nord, IFR Jean Roche, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France³

Received 28 December 2004/ Returned for modification 17 January 2005/ Accepted 17 May 2005

Peptidylglycine -amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal -amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

R.M. and L.O. are joint senior authors of the article.