Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogs of mRNA 5'-cap: changes in N7 substituent affect analog activity

Abstract

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

Novel “anti-reverse” cap analogs with superior translational properties

“…Similarly, mRNAs containing the bulky benzyl group on N7 (bn 7 Gp 3 G) instead of methyl are translated 1.7-fold more efficiently than mRNAs capped with the natural structure (Darzynkiewicz et al 1989). It is possible that m 2 2,7 Gp 3 G and bn 7 Gp 3 G behave as ARCAs, although we have not tested this.…”

“…Synthetic cap analogs have played an important role in elucidating these physiological processes. Studies on the inhibition of protein synthesis by cap analogs modified in the m 7 G, ribose, or phosphate moieties delineated the structural requirements for cap interactions with the cap-binding protein eIF4E (Adams et al 1978;Darzynkiewicz et al 1985Darzynkiewicz et al , 1987Darzynkiewicz et al , 1989. The conclusions drawn from these studies were further validated when the tertiary structure of eIF4E bound to m differs from that of eIF4E (Izaurralde et al 1994).…”

Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency

“…1). These represented the combination of four modifications that were previously shown to produce cap analogs with superior translational properties: benzyl-for-methyl substitution at N7 (Darzynkiewicz et al 1989), methoxy-for-hydroxyl substitution at the 3Ј-O position of the first Guo moiety (R 3 ; Stepinski et al 2001), addition of one methyl group at N2 of the same Guo moiety (R 1 ;Cai et al 1999), and addition of a fourth phosphate moiety (n = 2; Jemielity et al 2003). Chemical synthesis of the new cap analogs was performed by a strategy similar to that developed previously (Stepinski et al 2001;Jemielity et al 2003).…”

“…These have played important roles in elucidating splicing, intracellular transport, translation, and turnover, both as competitive inhibitors and as alternative structures at the 5Ј ends of RNAs. Some analogs are more inhibitory for translation than the corresponding natural compounds (Darzynkiewicz et al 1985(Darzynkiewicz et al , 1987(Darzynkiewicz et al , 1989 Gp 4 G (Cai et al 1999). The greatest inhibition is observed with cap analogs in the dinucleoside tetra-and pentaphosphate series (Cai et al 1999;Jemielity et al 2003).…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…To date, almost every position of m 7 G and TMG cap structures has been chemically modified, and the influence of those modifications on interaction with cap-binding proteins has been studied, often followed by the determination of the biological consequences of such modifications on RNA stability, transport, and translation. Interestingly, triphosphate chain modifications were explored much later than modifications of the 7-methylguanine or ribose moieties [36, [73][74][75][76], but turned out to have great potential to modulate the interactions of the cap-structure with cap-binding proteins and its susceptibility to different decapping enzymes.…”