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PubMed1982

Synthesis of mRNA by the parvovirus KRV in isolated nuclei.

We have synthesized vial-specific transcripts in nuclei isolated from cells infected with the parvovirus Kilham rat virus. Radioactive vial-specific RNA synthesized in the nuclei was extracted, hybridized to viral DNA, incubated in glyoxal, and electrophoresed in an agarose gel. At least five viral-specific RNAs were detected containing 4.6 Kb (25-26S), 3.2 Kb (major species, 20-22S), 2.9 Kb, 1.3 Kb, and 1.0 Kb pairs. The 4.6 Kb RNA represents a transcript of 95-100% of the viral genome. The major 3.2 and 2.9 Kb RNAs represent a transcript of 50-60% of the viral genome. Over 65% of the viral-specific RNA in the isolated nuclei is polyadenylylated on the 3' terminus. The 5' terminus of the RNA is capped in vitro by the sequence m7G(5')ppp(5')A. Incorporation of [beta-32p]ATP into the 5' cap sequence suggests that initiation of viral RNA synthesis may occur in the infected isolated nuclei.

Cell Nucleus↗
PubMed1981

Process of cap formation of messenger RNA by vaccinia virus particles carrying an organized enzymes system.

Vaccinia virus mRNAs carry the cap structure m7G5'pppAm- or m7G5'pppGm- at the 5'-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle. The process of the cap formation at the 5'-terminus of mRNA was studied using an in vitro system under similar conditions to those of vaccinia virus multiplication in its host cell. After adding a methyl-group donor, methyl-3H-S-adenosylmethionine, the oligonucleotides, which were the de novo synthesized 5'-terminal part of mRNA, were isolated from the RNA-synthesizing virion at appropriate time intervals, and were analysed. The 5'-5' confronting nucleotides with 2'-O-methylation, G5'pppAm and G5'pppGm, were found with the completed cap structure, m7G5'pppAm and m7G5'pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7G5'pppA and m7G5'pppG, were not detected at any incubation time, and it was concluded that methylation at the 2'-position of the 5'-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus (Moss et al., 1976) and also from the results obtained using other kinds of virus particles, which carry RNA polymerase and capping enzymes. These differences may be due to the specific organization of a series of capping enzymes and RNA polymerase in each virus particle.

Methylation↗
PubMed2002

Minimum molecular architectures for transcription and replication of the influenza virus.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.

Base Sequence↗
PubMed1998

RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein.

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.

Acid Anhydride Hydrolases↗
PubMed2001

HIV-1 Tat protein interacts with mammalian capping enzyme and stimulates capping of TAR RNA.

HIV gene expression is subject to a transcriptional checkpoint, whereby negative transcription elongation factors induce an elongation block that is overcome by HIV Tat protein in conjunction with P-TEFb. P-TEFb is a cyclin-dependent kinase that catalyzes Tat-dependent phosphorylation of Ser-5 of the Pol II C-terminal domain (CTD). Ser-5 phosphorylation confers on the CTD the ability to recruit the mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. Here we show that Tat spearheads a second and novel pathway of capping enzyme recruitment and activation via a direct physical interaction between the C-terminal domain of Tat and Mce1. Tat stimulates the guanylyltransferase and triphosphatase activities of Mce1 and thereby enhances the otherwise low efficiency of cap formation on a TAR stem-loop RNA. Our findings suggest that multiple mechanisms exist for coupling transcription elongation and mRNA processing.

Animals↗
PubMed1985

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N.

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.

Antibodies, Antinuclear↗
PubMed2003

Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase.

The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin. In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA. The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions. Pct1 did not interact with the S. pombe Ssn6 ortholog. In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein. Similarly, the S. cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene. The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins. Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression. These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.

Acid Anhydride Hydrolases↗
PubMed1988

A selective recognition mode of a nucleic acid base by an aromatic amino acid: L-phenylalanine-7-methylguanosine 5'-monophosphate stacking interaction.

The conformation of 7-methylguanosine 5'-monophosphate (m7GMP) and its interaction with L-phenylalanine (Phe) have been investigated by X-ray crystallographic, 1H-nuclear magnetic resonance, and energy calculation methods. The N(7) methylation of the guanine base shifts m7GMP toward an anti--gauche, gauche conformation about the glycosyl and exocyclic C(4')-C(5') bonds, respectively. The prominent stacking observed between the benzene ring of Phe and guanine base of m7GMP is primarily due to the N(7) guarternization of the guanine base. The formation of a hydrogen bonding pair between the anionic carboxyl group and the guanine base further stabilizes this stacking interaction. The present results imply the importance of aromatic amino acids as a hallmark for the selective recognition of a nucleic acid base.

Crystallography↗
PubMed1989

The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine.

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.

Binding Sites↗
PubMed1990

Fluorometric analysis of the long-wavelength absorption band of N-7 methylated GMP into the constituent bands of the two electronic states.

Steady-state measurements of fluorescence anisotropy are used to resolve the long-wavelength absorption spectrum of 7-methyl guanosine 5'-monophosphate (GMP) in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle theta between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on theta. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and approximately 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their CD spectra is discussed.

Fluorescence Polarization↗
PubMed1984

Chemical carcinogenesis. II. Imidazole-ring-opened derivatives from 7-ethyl- and 1,7-diethylguanosine.

The UV-spectral and chromatographic analyses of the derivatives of the two synthetic standards 7-ethylguanosine and 1,7-diethylguanosine are here reported. The derivatives obtained from the dialkyl compound exhibit a striking similarity to those found in the "pyrimidine-nucleotide-like" fraction of rat liver tRNA ethylated in vivo by ethionine. The finding of imidazole-ring-opened products in tRNA ethylation by ethionine could be significant from the point of view of chemical carcinogenesis: in fact, imidazole-ring-opening of 1,7-dialkylguanosines directly at level of RNA with consequent formation of substituted pyrimidines is a transversion, i.e. a mutagenic event which would cause a change in the expression of genetic information since a purine has been transformed into a pyrimidine.

Animals↗
PubMed2002

The yeast capping enzyme represses RNA polymerase II transcription.

Using a highly pure transcription system derived from Saccharomyces cerevisiae, we have purified an activity in yeast whole-cell extracts that represses RNA polymerase II transcription. Mechanistic studies suggest that this repressor specifically targets transcriptional reinitiation. The two polypeptides that constitute the repressor have been identified as Ceg1p and Cet1p, the two subunits of the yeast pre-mRNA capping enzyme. A purified recombinant capping enzyme is able to reconstitute repressor activity. Cet1p is necessary for and capable of this repression. Transcriptional run-on experiments indicate that the capping enzyme also serves as a repressor in vivo. Efficient pre-mRNA capping relies on interactions between the capping enzyme and transcription apparatus. Repression by the capping enzyme suggests a bidirectional flow of information between capping and transcription.

Acid Anhydride Hydrolases↗
PubMed2002

Unique capping activity of the recombinant RNA polymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein.

Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5'-ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp(alpha)p(beta)-p(alpha)A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction.

Animals↗
PubMed1987

Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA.

The mini-exon, a short segment found at the 5' end of trypanosome mRNAs, is contributed by a small RNA, the mini-exon donor (medRNA). In vivo 32P-labeled medRNA, a set of smaller RNAs related to it, and mRNA, were purified from Trypanosoma brucei by hybrid selection and gel electrophoresis. Using RNA fingerprinting and sequencing techniques, mini-exon oligonucleotides were identified and characterized. We detected a novel 5' terminal capped oligonucleotide present in both medRNA and mRNA. This structure contained m7G and at least four modified nucleotides, not identified previously. If the T. brucei mini-exon has exactly four transcribed nucleotides upstream from its originally designated 5' end, it would begin with the sequence: m7GpppA*A*C*U*AA*CG (asterisks denote modification) and medRNA would be 140 nucleotides long, excluding the m7G residue. The mini-exon contains, and retains during its transfer to mRNA, a novel 5' terminal structure whose presence could confer unique functional attributes.

Animals↗
PubMed1986

Stacking and hydrogen bonding interactions between phenylalanine and guanine nucleotide: crystal structure of L-phenylalanine-7-methylguanosine-5'-monophosphate complex.

The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein.

Carrier Proteins↗
PubMed1979

The 5' terminal capping of heterogeneous nuclear RNA at different embryonic stages of the sea urchin.

5' Terminal cap structures of hnRNA have been characterized and the extent of capping determined as a function of embryonic development. Sea urchin embryo hnRNA contains only the type-1 cap, m7GpppNmpNp, with the type-2 cap, which has a 2'-0-methylated subpenultimate nucleotide, being associated only with stable small nuclear RNAs. These cap 2-containing RNAs are synthesized at a rate of approximately 70 molecules min-1 nucleus-1 compared to approximately 1000 molecules for hnRNA cap 1. Approximately 70% of nuclear cap 1 is associated with greater than 15S RNA in denaturing solvent, but under non-denaturing conditions the percentage is much higher. Cap 1 in low and high molecular weight nuclear RNA have the same kinetics of methyl labeling. Thus all cap 1 structures may belong to a single class either covalent or H-bonded to high molecular weight RNA. hnRNA greater than 15S is 35% capped; however, adding caps in less than 15S RNA gives an estimate of 50% capping for total hnRNA. In development from early blastula to late gastrula, there is little if any change in the extent of capping of hnRNA. These results coupled with others indicate that the fraction of hnRNA molecules serving as precursor to mRNA does not change quantitatively during embryonic development.

Animals↗
PubMed2006

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA.

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.

3' Untranslated Regions↗