Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps (original) (raw)
Related papers
Biochemistry, 1987
New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of-0 with-H,-CH3, or-NH2. Additional analogues were synthesized with &methyl-or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 'H and 31P N M R for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by-H and-CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by-NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the a-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation. T a n s l a t i o n of eukaryotic mRNA is dependent on several aspects of mRNA structure. These macromolecules are for the most part monocistronic, possess 5'-termini of the form m7G(5')ppp(5')N, and are polyadenylylated on the 3'-end. The 5' modification is known as a cap and is necessary for optimum translation. It is recognized by at least three translation initiation factors, namely, eIF-4A,' eIF-4B, and eIF-4F (Grifo et al., 1983; Edery et al., 1983, 1985). These are also collectively termed cap binding proteins (CBPs; Shatkin, 1985). These factors bind at or near the 5'-cap of mRNA early in the initiation phase of protein biosynthesis and facilitate mRNA attachment to the 40s ribosomal subunit. Cap analogues inhibit this step of initiation both in complete translation assay (
Biochemistry, 1989
New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -0 with -H, -CH3, or -NH2. Additional analogues were synthesized with &methyl-or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 'H and 31P N M R for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the a-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.
Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.
Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA
International Journal of Molecular Sciences, 2019
Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5 untranslated regions (UTRs). Scanning through the 5 UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5 UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5 UTR but not that of an mRNA with a poly(A) sequence as the 5 UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5-triphosphate (AMP-PNP) inhibited translation irrespective of the 5 UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA ('toeprinting'), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This '−8 nt toeprint' was seen with mRNA 5 UTRs of different length, sequence and structure potential. Importantly, the '−8 nt toeprint' was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5 UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5 UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.
RNA, 2005
The eukaryotic initiation factor eIF4E binds the mRNA 5 0 cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4Emediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m 7 GTP and m 7 GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.
Proceedings of the National Academy of Sciences, 1977
ABSTRACr Vaccinia mRNAs containing either 5'-terminal m7G or unmethylated 5'-terminal structures were synthesized in vitro and their relative efficiencies of translation were compared in wheat germ and reticulocyte cell-free proteinsynthesizing systems. The importance of the m7G group for efficient translation increases as the K+ concentration is raised. At K+ concentrations optimal for translation of mRNA containing m7G, unmethylated mRNA is translated at the same relative low efficiency in both cell extracts. The rate of binding of mRNA to ribosomes at K+ concentrations close to those found in intact cells is strongly influenced by the presence of m7G regardless of the source of the cell extract. Experiments designed to test the function of the 5'-terminal m7G(5')ppp cap structure of eukaryotic mRNAs have shown that either chemically removing the 5'-terminal m7G or preventing the methylation involved in its formation reduces the ability of mRNAs to be translated in vitro (1). However, a low level of translation of these mRNAs remains in all cases, and the extent of this residual translation varies considerably when tested in different cell-free systems (1-6). It has been suggested that this variability might be due to characteristics of the protein-synthesizing machinery of different cell types (4-6), and to differences in the interaction between mRNA structures other than the 5'-terminal m7G and components of the cell-free system (5). In view of these results, the role of 5'-terminal m7G in mRNA translation has recently been questioned (7). The experiments reported here show that the varying dependency on the 5'-terminal m7G for translation of eukaryotic mRNAs in vitro can be the result of the conditions introduced during the assembly of the cell-free systems. We have examined the relative translational efficiency of the methylated and unmethylated forms of vaccinia virus mRNA over a range of K+ concentrations in cell-free protein-synthesizing systems derived either from wheat germ or from rabbit reticulocytes. Our results show that the importance of the 5'-terminal m7G for mRNA translation increases as the K+ concentration in the cell-free system is increased, regardless of the source of the cell extract. By using KOAc rather than KCI, protein synthesis can be efficiently carried out in vitro at K+ concentrations close to those found in intact cells (34). At these K+ concentrations unmethylated mRNA is translated at 15-20% the efficiency of methylated mRNA in both reticulocyte and wheat germ cell-free systems. The difference in the relative translation of methylated and unmethylated mRNA results from a reduced rate of binding of unmethylated mRNA to ribosomes. MATERIALS AND METHODS Chemicals. pm7G was purchased from P-L Biochemicals. Sparsomycin was a gift from Upjohn Co. Radioactive compounds were purchased from New England Nuclear. Synthesis, Purification, and Characterization of Vaccinia Virus mRNA. Purified vaccinia virus (stain WR) was a gift of Enzo Paoletti, of the New York State Health Laboratories. The vaccinia virus was prepared from infected HeLa cells as described by Joklik (8). RNA was synthesized in vitro in reaction
General RNA binding proteins render translation cap dependent
The EMBO journal, 1996
Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start si...