Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen.
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.
Brenet
Fabienne
F
Université de la Méditerranée, Aix-Marseille II, Laboratoire de Cancérologie Expérimentale, Inserm EMI 0359, Faculté de Médecine Secteur Nord, IFR Jean Roche, Marseille, France.
Dussault
Nadège
N
Borch
Jonas
J
Ferracci
Géraldine
G
Delfino
Christine
C
Roepstorff
Peter
P
Miquelis
Raymond
R
Ouafik
L'Houcine
L
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D011072:Poly U / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D000097585:SS-B Antigen
D001324:Autoantigens; D008024:Ligands; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D011072:Poly U; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; C035189:peptidylglycine monooxygenase