5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.     

Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development

Up to the midblastula stage, cell division in Xenopus is directed by maternal mRNAs and proteins that are inherited by the fertilized egg. We have isolated cDNA clones for mRNAs that undergo either adenylation or deadenylation during this developmental period. Coincidental with this poly(A) metabolism are changes in polysomal localization. The relationship between polyadenylation and translation is discussed.     

Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

The 5′-Nucleotidases and Cyclic Phosphodiesterases (3′-Nucleotidases) of the Enterobacteriaceae

All members of the Enterobacteriaceae possess distinct 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) that can be differentiated from the acid and alkaline phosphatases and the acid sugar hydrolases. The nucleotidases and cyclic phosphodiesterases of the various Enterobacteriaceae are remarkably similar in properties. All of the 5'-nucleotidases hydrolyze 5'-nucleotides, adenosine triphosphate, and uridine diphosphoglucose. Their pH optimum is from 5.7 to 6.1. The cyclic phosphodiesterases hydrolyze 3'-nucleotides, cyclic phosphonucleotides, bis-(p-nitrophenyl)phosphate, and p-nitrophenylphosphate. Their pH optimum is from 7.2 to 7.8. For both enzymes, cobalt showed optimal metal stimulation. An intracellular protein inhibitor for the 5'-nucleotidase is present in all of the Enterobacteriaceae. No inhibitor of cyclic phosphodiesterase activity exists, although hydrolysis of both cyclic phosphonucleotides and 3'-nucleotides is inhibited by ribonucleic acid. Neither of the enzymes is subject to control by phosphate level or by catabolite repression. Of the other bacteria studied, only Haemophilus and Bacillus subtilis contained significant 3'- or 5'-nucleotidase activity.     

Expression of Neurotransmitter Receptors by mRNAs from Neurons Developing In Vitro: A Xenopus Oocyte Expression Study

Abstract: Poly(A)⁺ mRNA was extracted from cultures of neurons isolated from mouse embryonic day 14 cerebral cortex and injected into Xenopus oocytes. This led to the expression of receptors for γ-aminobutyric acid (GABA), glycine, acetylcholine, serotonin, glutamate, kainate, N -methyl-D-aspartate, and quisqualate. Northern blot analysis of poly(A)⁺ mRNA from the cultured neurons with a GluRI cDNA probe revealed the presence of three hybridization bands with estimated mRNA sizes of 5.1, 4.0, and 3.1 kb, respectively. The development of mRNAs coding for neurotransmitter receptors was investigated by isolating mRNA from neurons cultured for 2, 8, and 14 days in vitro and injecting it into Xenopus oocytes. The amplitude of membrane currents elicited by the transmitters gave a measure of the relative amounts of the different mRNAs. The size of the responses to kainate, aspartate (together with glycine), glutamate, acetylcholine, GABA, serotonin, and glycine increased with the time of culture in vitro. However, in contrast to all other agonist-induced currents, the current induced by glycine failed to increase further from 8 to 14 days in culture. It is concluded that the time course of receptor development in cortical neurons in vitro is similar to the development in vivo.     

7-Deaza-2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3′- and 5′-junctions of d(A6)·d(T6) and B-DNA

7-Deaza-2′-deoxyadenosine (1, c⁷A_d) and 3-deaza-2′-deoxyadenosine (2, c³A_d) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthsized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5′ by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c³A_d decreased the bending more than replacement by c⁷A_d. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3′-site of an d(AAAAAA)-tract whereas replacement at the 5′-site showed no significant influence [1, 2].     

Translation-Competent Extracts fromSaccharomyces cerevisiae:Effects of L-A RNA, 5′ Cap, and 3′ Poly(A) Tail on Translational Efficiency of mRNAs

Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitablein vitrotranslation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-freeSaccharomyces cerevisiaelysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5′ cap and a 3′ poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events.     

Regulation of Coronaviral Poly(A) Tail Length during Infection

The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation, in vivo, of RNAs with the longer poly(A) tail was enhanced over those with the shorter poly(A). Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels.     

Heat Shock Disrupts Cap and Poly(A) Tail Function during Translation and Increases mRNA Stability of Introduced Reporter mRNA

The effect of heat shock on translational efficiency and message stability of a reporter mRNA was examined in carrot (Daucus carota). Heat shock of short duration resulted in an increase in protein yield, whereas repression was observed following extended exposure to the stress. Regardless of the duration of the heat shock, a loss in the function of the 5[prime] cap [m7G(5[prime])ppp(5[prime])N, where N represents any nucleotide] and the 3[prime] poly(A) tail, two regulatory elements that work in concert to establish an efficient level of translation, was observed. This apparent paradox was resolved upon examination of the mRNA half-life following thermal stress, in which increases up to 10-fold were observed. Message stability increased as a function of the severity of the heat shock so that following a mild to moderate stress the increase in message stability more than compensated for the reduction in cap and poly(A) tail function. Following a severe heat shock, the increased mRNA half-life was not sufficient to overcome the virtual loss in cap and poly(A) tail function. No stimulation of protein synthesis was observed following a heat shock in Chinese hamster ovary cells, data suggesting that the heat-induced increases in mRNA stability may be unique to the heat-shock response in plants.     

Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p

The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

The Antibacterial Properties of 4, 8, 4′, 8′-Tetramethoxy (1,1′-biphenanthrene) -2,7,2′,7′-Tetrol from Fibrous Roots of Bletilla striata

Biphenanthrene compound, 4, 8, 4′, 8′-tetramethoxy (1, 1′-biphenanthrene)—2, 7, 2′, 7′-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.     

In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.     

NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP⁺ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.