Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity.
Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins
Darzynkiewicz
E
E
Department of Microbiology, University of Southern California School of Medicine, Los Angeles 90033-1054.
Stepinski
J
J
Ekiel
I
I
Goyer
C
C
Sonenberg
N
N
Temeriusz
A
A
Jin
Y
Y
Sijuwade
T
T
Haber
D
D
Tahara
S M
SM
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D014176:Protein Biosynthesis / Q000187:drug effects*; D012314:RNA Cap Analogs / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D012314:RNA Cap Analogs; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D006157:Guanosine Monophosphate