Removal of 5''-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation.

The procedure for isolation of nucleotide pyrophosphatase (E.C. 3.6.1.9.) from potato has been modified to yield an endonuclease-free preparation purified 2300-fold. The enzyme was used for specific cleavage of pyrophosphate linkages in the 5'-terminal cap (m7GpppN) of several eukaryotic messenger RNAs. Enzymatic removal of 5'-terminal pm7G from reovirus, rabbit globin and Artemia salina mRNAs resulted in an almost complete loss (greater than 80%) of their template activities in a cell-free protein synthesizing system from wheat germ. Incubation with nucleotide pyrophosphatase did not decrease the translation of phage f2 RNA in an Escherichia coli cell-free system.     

Requirement for 7-methylguanosine in translation of globin mRNA in vivo.

The 7-methylguanosine (m7G) residue present in the m7G5' ppp5'X-"CAP" structure of rabbit globin mRNA was removed quantitatively by periodate oxidation followed by beta-elimination in the presence of cyclohexylamine. The RNA thus treated was intact and exhibited no signs of degradation as examined by polyacrylamide gel electrophoresis in formamide. Assay for protein synthesis using a wheat germ cell-free system showed that the globin mRNA lacking m7G had lost most of its messenger activity. Identical treatment, of satellite tobacco necrosis virus (STNV) RNA, which does not contain the 5'-terminal "CAP" structure, resulted in no loss of its mRNA activity. Since the importance of the m7G residue in eukaryotic mRNA has not yet been shown essential for translation in vivo, both untreated and treated globin mRNAs were injected into frog oocytes and their translation into globin was measured at intervals over a ninety-six hour period. Globin mRNA either treated with periodate alone or lacking in m7g altogether were both found to have lost more than 90% of their activity in vivo.     

Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

In vitro translation of the intermediate filament proteins desmin and vimentin.

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.     

Auxin- and ethylene-induced changes in the population of translatable messenger RNA in Basal sections and intact soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated basal sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. Within one hour of 2,4-dichlorophenoxyacetic acid treatment, the translatable messenger RNAs for at least twelve in vitro translation products are modulated upward. In vitro translation products of polyadenylated RNA from untreated, auxin-treated and Ethephon-treated intact soybean hypocotyl were also analyzed. Within two hours of treatment with either 2,4-dichlorophenoxyacetic acid or Ethephon, the translatable messenger RNAs for a group of high molecular weight in vitro translation products are modulated upward. There is a particular set of translatable messenger RNA, encoding in vitro translation products in the 24,000 to 32,000 molecular weight range, that is specifically modulated upward by auxin treatment in intact soybean hypocotyl and in hypocotyl sections.     

Reverse 5' caps in RNAs made in vitro by phage RNA polymerases.

We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.     

The Nucleic Acid-binding Domain and Translational Repression Activity of a Xenopus Terminal Uridylyl Transferase

Terminal uridylyl transferases (TUTs) catalyze the addition of uridines to the 3′ ends of RNAs and are implicated in the regulation of both messenger RNAs and microRNAs. To better understand how TUTs add uridines to RNAs, we focused on a putative TUT from Xenopus laevis, XTUT7. We determined that XTUT7 catalyzed the addition of uridines to RNAs. Mutational analysis revealed that a truncated XTUT7 enzyme, which contained solely the nucleotidyl transferase and poly(A) polymerase-associated domains, was sufficient for catalytic activity. XTUT7 activity decreased upon removal of the CCHC zinc finger domains and a short segment of basic amino acids (the basic region). This basic region bound nucleic acids in vitro. We also demonstrated that XTUT7 repressed translation of a polyadenylated RNA, to which it added a distinct number of uridines. We generated a predicted structure of the XTUT7 catalytic core that indicated histidine 1269 was likely important for uridine specificity. Indeed, mutation of histidine 1269 broadened the nucleotide specificity of XTUT7 and abolished XTUT7-dependent translational repression. Our data reveal key aspects of how XTUT7 adds uridines to RNAs, highlight the role of the basic region, illustrate that XTUT7 can repress translation, and identify an amino acid important for uridine specificity.     

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     

Synergistic effects of eIF4A and MEK inhibitors on proliferation of NRAS-mutant melanoma cell lines

Activating mutations of the NRAS (neuroblastoma rat sarcoma viral oncogene) protein kinase, present in many cancers, induce a constitutive activation of both the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway and the PI(3)K-AKT-mTOR, pathway. This in turn regulates the formation of the eIF4F eukaryotic translation initiation complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, which binds to the 7-methylguanylate cap (m(7)G) at the 5′ end of messenger RNAs. Small molecules targeting MEK (MEKi: MEK inhibitors) have demonstrated activity in NRAS-mutant cell lines and tumors, but resistance sets in most cases within months of treatment. Using proximity ligation assays, that allows visualization of the binding of eIF4E to the scaffold protein eIF4G, generating the active eIF4F complex, we have found that resistance to MEKi is associated with the persistent formation of the eIF4F complex in MEKi-treated NRAS-mutant cell lines. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a small molecule of the flavagline/rocaglate family, synergizes with inhibiting MEK to kill NRAS-mutant cancer cell lines.     

Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.     

Chemical route to the capped RNAs

Eukaryotic and viral messenger RNAs contain a CAP structure that plays an important role in the initiation of translation and several other cellular processes that involve mRNAs. In this paper, we report a convenient chemical approach to the preparation of milligram quantities of short, capped RNA oligonucleotides, which overcomes some of the limitations of previous approaches. The method is based on the use of a reactive precursor, m7GppQ [P1-7-methylguanosine-5'-O-yl, P2-O-8-(5-chloroquinolyl) pyrophosphate]. The precursor reacts smoothly with 5'-phosphorylated unprotected short RNA in the presence of CuCl2 in organic media. The feasibility of this approach was demonstrated by the synthesis of the capped pentaribonucleotide m7GpppGpApCpU. The synthesized capped oligonucleotide was isolated and purified by reverse phase and ion exchange HPLC with a final yield of 37%. The structure of the m7GpppGpApCpU was confirmed by 31P NMR, mass-spectrometry and enzymatic hydrolysis.     

Intrinsic RNA Binding by the Eukaryotic Initiation Factor 4F Depends on a Minimal RNA Length but Not on the m7G Cap

The eukaryotic initiation factor 4F (eIF4F) is thought to be the first factor to bind mRNA during 7-methylguanosine (m⁷G) cap-dependent translation initiation. The multipartite eIF4F contains the cap-binding protein eIF4E, and it is assumed that eIF4F binds mRNAs primarily at the 5′ m⁷G cap structure. We have analyzed equilibrium binding of rabbit eIF4F to a series of diverse RNAs and found no impact of the 5′-cap on the stability of eIF4F-RNA complexes. However, eIF4F preferentially and cooperatively binds to RNAs with a minimum length of ∼60 nucleotides in vitro. Furthermore, translation activity in rabbit reticulocyte lysate is strongly inhibited by RNAs exceeding this length, but not by shorter ones, consistent with the notion that eIF4F in its physiological environment preferentially binds longer RNAs, too. Collectively, our results indicate that intrinsic RNA binding by eIF4F depends on a minimal RNA length, rather than on cap recognition. The nonetheless essential m⁷G cap may either function at steps subsequent to eIF4F-RNA binding, or other factors facilitate preferential binding of eIF4F to the m⁷G cap.     

RNA degradation in Saccharomyces cerevisae

All RNA species in yeast cells are subject to turnover. Work over the past 20 years has defined degradation mechanisms for messenger RNAs, transfer RNAs, ribosomal RNAs, and noncoding RNAs. In addition, numerous quality control mechanisms that target aberrant RNAs have been identified. Generally, each decay mechanism contains factors that funnel RNA substrates to abundant exo- and/or endonucleases. Key issues for future work include determining the mechanisms that control the specificity of RNA degradation and how RNA degradation processes interact with translation, RNA transport, and other cellular processes.