Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

The methyl ester of m⁷G^5′ p was synthesized by a carbodiimide-catalyzed reaction of G^5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m⁷Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m⁷G^5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m⁷G^5′ p than to m⁷Gp · methyl ester. Conversion of the esterified analog to m⁷G^5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m⁷G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.     

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.     

Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene.

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M·Eco P1) or with the DNA-adenine methylase (M·Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M·Eco dam modification of calf thymus DNA. The results show that M·Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M·Eco P1 has also been deduced; we propose that M·Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A¹-C-Py … 3′ (where $\text{A}{}^1\text{= N}{}^6$ methyladenine and Py is C or T).     

Chemical synthesis of capped RNA fragments and their ability to complex with eukaryotic ribosomes

In other to examine the binding ability of the 5'-terminal part of eukaryotic mRNA to 80S ribosome, several kinds of oligoribonucleotides, pA-U-G, m7G5'pppA-U-G, m7G5'pppG-U, m7G5'pppA-U-G-A-C-C, were synthesized chemically. The binding experiments of oligonucleotides to 80S ribosome showed that the capped structure as well as AUG are essential for ribosome binding, and the efficiency is enhanced by the 5'-leader sequence if it would include complementary sequence to the 3'-terminal part of 18S rRNA.     

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

In vitro synthesis of a possible precursor RNA by purified vesicular stomatitis virus.

The RNA products synthesized $\text{in vitro}$ by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA $\text{in vitro}$.     

Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

Oligonucleotide analogues containing internucleotide C3′-CH<Subscript>2</Subscript>-C(O)-NH-C5′ bonds

A dinucleoside bearing an amide internucleotide C3′-CH₂-C(O)-NH-C5′ bond was synthesized by the interaction of 3′-deoxy-3′-carboxylmethylribothymidine-2′,3′-lactone obtained by hydrolysis of 2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-ethoxycarboxylmethylribothymidine with 5′-deoxy-5′-amino-3′-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3′-O-(2-cyanoethyl-N,N′-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones.     

Sequence specificity of the wild-type dam+) and mutant (damh) forms of bacteriophage T2 DNA adenine methylase

Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam⁺) or mutant (dam^h) T2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by two-dimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive N⁶-methyldeoxyadenosine. From this analysis we conclude that both T2 dam⁺ and T2 dam^h contain the sequence 5′…G-mA -Py…3′.