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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 {alpha}-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,{dagger} and L'Houcine Ouafik1,{dagger}*

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 {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal {alpha}-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.

* Corresponding author. Mailing address: Inserm EMI 0359, Faculté de Médecine Secteur Nord, IFR Jean Roche, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France. Phone: (33) 491698740. Fax: (33) 491090171. E-mail: ouafik.h{at}jean-roche.univ-mrs.fr.

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

Molecular and Cellular Biology, September 2005, p. 7505-7521, Vol. 25, No. 17
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.17.7505-7521.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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