Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2′-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2′-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2′-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.     

The sequence and secondary structure of the 3′-UTR affect 3′-end maturation,RNA accumulation,and translation in tobacco chloroplasts

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5- and 3-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3-UTR also influenced -glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3-UTR may influence translational yield.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Leeches as a source of mammalian viral DNA and RNA—a study in medicinal leeches

Surveillance of wild vertebrates can be challenging in remote and inaccessible areas such as tropical rainforests. Blood-feeding parasites, such as leeches, can facilitate wild vertebrate monitoring by targeting residual DNA from the animals the leeches feed on. Successes in detecting host DNA from leeches suggest that host viruses may also be detectable. To systematically test this hypothesis, we performed a proof of concept study using quantitative PCR (qPCR) to detect DNA viruses (bovine herpesvirus [BHV], human adenovirus [HAdV]) and RNA viruses (influenza A [InfA] and measles morbillivirus [MeV]) from nucleic acids extracted from medicinal leeches fed with blood spiked with each virus. All viruses except BHV showed a gradual decline in concentration from day 1 to 50, and all except BHV were detectable in at least half of the samples even after 50 days. BHV exhibited a rapid decline at day 27 and was undetectable at day 50. Our findings in medicinal leeches indicate that leeches collected in the wild might be an untapped resource for detecting vertebrate viruses and could provide new opportunities to study wildlife viral diseases of rare species in challenging environments, where capturing and handling of animals is difficult.     

Differential expression of 2′∶3′-cyclic nucleotide 3′-phosphodiesterase in cultured central,peripheral, and extraneural cells

The relative levels of the central nervous system myelin marker enzyme 2:3-cyclic nucleotide 3-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C₆ and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.     

The role of zinc in 5′,5′-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases

Summary Aminoacyl-tRNA synthetases are capable of converting 5-ATP into 5,5-diadenosine tetraphosphate. The reaction reflects the reversal of enzyme-bound aminoacyl-adenylate by ATP instead of PP_i.In the case of a few prokaryotic as well as eukaryotic aminoacyl-tRNA synthetases, the initial rate of diadenosine tetraphosphate synthesis can be greatly enhanced upon adding small amounts of zinc. This observation enables us to establish a relationship between diadenosine tetraphosphate, a nucleotide possibly involved in controlling cell proliferation, and a metallic cofactor, which is believed to play a role in tumour growth.Abbreviations AlaRS alanyl-tRNA synthetase (EC 6.1.7) - CysRS cysteinyl-tRNA synthetase (EC 6.1.16) - HisRS histidyl-tRNA synthetase (EC 6.1.21) - HeRS isoleucyl-tRNA synthetase (EC 6.1.5) - LysRS lysyl-tRNA synthetase (EC 6.1.6) - MetRS methionyl-tRNA synthetase (EC 6.1.10) - PheRS phenylalanyl-tRNA synthetase (EC 6.1.20) - ProRS prolyl-tRNA synthetase (EC 6.1.15) - TrpRS Tryptophanyl-tRNA synthetase (EC 6.1.2) - TyrRS tyrosyl-tRNA synthetase (EC 6.1.1) - EDTA ethylene diamine tetraacetic acid     

RNA recombination in turnip crinkle virus: its role in formation of chimeric RNAs,multimers, and in 3′-end repair

RNA recombination is well documented for an increasing number of viruses and it is thought to have affected viral evolution and adaptation. However, molecular mechanisms mediating crossover events have been studied in only a few viral systems due to difficulties such as the low frequency of the event, and the scattered distribution of junction sites. Therefore, the uniquely high recombination frequency and nonrandom crossover site distribution for recombination among turnip crinkle carmovirus (TCV) RNAs make TCV an excellent model recombination system. Characterization of large numbers of junction sites between two species of TCV satellite RNAs and between the TCV genomic RNA and one of the satellite RNAs revealed for the first time the role of specific sequences and structures in recombination. Also, recombination was found to play a role in formation of novel chimeric RNAs, multimeric forms of satellite RNAs as well in 3′ end repair of mutated satellite RNAs. A replicase-driven template-switching model is presented to explain many common features occurring during recombination in TCV infections.     

The role of the anticodon in the interaction between methionyl-tRNA synthetase and bacterial initiator tRNA?

Complementary and antiparallel oligonucleotides bind to exposed regions of the tRNA molecule. Aminoacylation in the presence of triplets has been used to determine the role of the anticodon in the interaction between methionyl-tRNA synthetase and initiator tRNA. ApUpG has no effect on the charging even when 70% of the tRNA is bound to the triplet, whereas in the presence of GpGpU which binds to the A-C-C sequence adjacent to the 3' terminal adenosine that fraction of the tRNA which is bound to the triplet is completely unavailable for charging. Hence the anticodon is probably not involved in a primary interaction while the A-C-C-A-OH clearly is. This conclusion is supported by the failure of the isolated anticodon loop and stem oligonucleotides to inhibit the aminoacylation reaction.     

Immunohistochemical localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in adult bovine cerebrum and cerebellum

We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.     

The role of growth rate, redox-state of the plastoquinone pool and the trans-thylakoid ΔpH in photoacclimation of Chlorella vulgaris to growth irradiance and temperature

The long-term photoacclimation of Chlorella vulgaris Beijer (UTEX 265) to growth irradiance and growth temperature under ambient CO₂ conditions was examined. While cultures grew at a faster rate at 27 than at 5 °C, growth rates appeared to be independent of irradiance. Decreases in light-harvesting polypeptide accumulation, increases in xanthophyll pool size and changes in the epoxidation state of the xanthophyll cycle pigments were correlated linearly with increases in the relative reduction state of Q_A, the primary quinone receptor of photosystem II, when estimated as 1−q_P under steady-state growth conditions. However, we show that there is also a specific temperature-dependent component, in addition to the redox-state of the Q_A, involved in regulating the content and composition of light-harvesting complex II of C. vulgaris. In contrast, modulation of the epoxidation state of the xanthophyll pool in response to increased 1−q_P in cells grown at 5 °C was indistinguishable from that of cells grown at 27 °C, indicating that light and temperature interact in a similar way to regulate xanthophyll cycle activity in C. vulgaris. Because C. vulgaris exhibited a low-light phenotype in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but a high-light phenotype upon addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, we conclude that the plastoquinone pool acts as a sensor regulating the accumulation of light-harvesting polypeptides in C. vulgaris. However, concomitant measurements of non-photochemical fluorescence quenching (q_N) and the epoxidation state of the xanthophyll pool appear to indicate that, in addition to the redox-state of the plastoquinone pool, the trans-thylakoid ΔpH may also contribute to sensing changes in irradiance and temperature that would lead to over-excitation of the photosynthetic apparatus. We suggest that sink capacity as reflected in photosynthate utilization and cell growth ultimately regulate photoacclimation in C. vulgaris. Received: 17 April 2000 / Accepted: 23 May 2000     

Synthesis,characterization, and reactions of 2′-, 3′-, and 5′-(2-bromoethyl)-AMP: Potential affinity labels for nucleotide binding sites in enzymes

Two new adenosine analogs, 2′-(2-bromoethyl) adenosine monophosphate and 3′-(2-bromoethyl) adenosine monophosphate, were synthesized, purified by semipreparative high-pressure liquid chromatography, and completely characterized. A new synthesis of 5′-(2-bromoethyl) adenosine monophosphate is presented which facilitates the preparation of radioactive reagent with label either in the ethyl group or the purine ring of the nucleotide derivative. The reactive moiety of these derivatives, a bromoalkyl group, has the ability to react with the nucleophilic side chains of several amino acids. The second-order, pH-independent rate constants for reaction with the side chains of the amino acids cysteine, lysine, histidine, and tyrosine were determined as 3×10^?4, 6×10^?6, 3×10^?7, and <1×10^?7 M^?1 sec^?1, respectively. These data could be use in estimating the rate enhancement observed in modification of a protein by these affinity-labeling reagents. 5′-(S-(2-hydroxyethyl)cysteine) adenosine monophosphate, the derivative expected from exhaustive digestion of protein in which a cysteinyl residue is modified by 5′-(2-bromoethyl) adenosine monophosphate, and S-2-hydroxyethyl)cysteine, the derivative anticipated upon acid hydrolysis of such a modified protein, were synthesized, characterized, and their elution positions from an amino acid analyzer determined. These bromoethyl AMP derivatives are potential affinity labels for enzymes that bind 2′-, 3′-, or 5′-nucleotides such as TPN, coenzyme A, or ADP, respectively.     

Pathophysiological and molecular considerations of viral and bacterial infections during maternal-fetal and –neonatal interactions of SARS-CoV-2, Zika,and Mycoplasma infectious diseases

During pregnancy, a series of physiological changes are determined at the molecular, cellular and macroscopic level that make the mother and fetus more susceptible to certain viral and bacterial infections, especially the infections in this and the companion review. Particular situations increase susceptibility to infection in neonates. The enhanced susceptibility to certain infections increases the risk of developing particular diseases that can progress to become morbidly severe. For example, during the current pandemic caused by the SARS-CoV-2 virus, epidemiological studies have established that pregnant women with COVID-19 disease are more likely to be hospitalized. However, the risk for intensive care unit admission and mechanical ventilation is not increased compared with nonpregnant women. Although much remains unknown with this particular infection, the elevated risk of progression during pregnancy towards more severe manifestations of COVID-19 disease is not associated with an increased risk of death. In addition, the epidemiological data available in neonates suggest that their risk of acquiring COVID-19 is low compared with infants (<12 months of age). However, they might be at higher risk for progression to severe COVID-19 disease compared with older children. The data on clinical presentation and disease severity among neonates are limited and based on case reports and small case series. It is well documented the importance of the Zika virus infection as the main cause of several congenital anomalies and birth defects such as microcephaly, and also adverse pregnancy outcomes. Mycoplasma infections also increase adverse pregnancy outcomes. This review will focus on the molecular, pathophysiological and biophysical characteristics of the mother/placental-fetal/neonatal interactions and the possible mechanisms of these pathogens (SARS-CoV-2, ZIKV, and Mycoplasmas) for promoting disease at this level.     

Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Hen1 is an RNA ribose 2′-O-methyltransferase that modifies the 3′ terminal nucleoside of eukaryal small regulatory RNAs. Here, we report that Hen1 homologs are present in bacterial proteomes from eight different phyla. Bacterial Hen1 is encoded by the proximal ORF of a two-gene operon that also encodes polynucleotide kinase-phosphatase (Pnkp), an RNA repair enzyme. Purified recombinant Clostridium thermocellum Hen1 is a homodimer of a 465-amino acid polypeptide. CthHen1 catalyzes methyl transfer from AdoMet to the 3′ terminal nucleoside of an RNA oligonucleotide, but is unreactive with a synonymous DNA oligonucleotide or an RNA with a single 3′-terminal deoxyribose sugar. CthHen1 is optimally active at alkaline pH and dependent on manganese. Activity is inhibited by AdoHcy and abolished by mutations D291A and D316A in the putative AdoMet-binding pocket. The C-terminal fragment, Hen1-(259–465), comprises an autonomous monomeric methyltransferase domain.     

Structure and mechanism of the 2′,3′ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn²⁺-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn²⁺ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn²⁺ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.     

The role of melanocytes in protection against photooxidative stress Krystyna Stepień

Excessive exposure to solar ultraviolet radiation is an essential etiological factor for skin cancer. UV radiation, directly or indirectly through the generation of reactive oxygen species (ROS), causes damage to DNA, proteins and lipids, and induces inflammation and immunosuppression. Cutaneous pigmentation afforded by melanocytes is the main photoprotective mechanism in human skin. In response to UV, melanocytes produce melanin pigments and transfer them to adjacent keratinocytes. This review describes: (i) the photoprotective action of melanin; (ii) the regulation of UV-induced melanogenesis and the role of p53 in this process; (iii) the relation between melanogenic and antioxidant activities in melanocytes. The possible involvement of UV-induced ROS in the stimulation of melanin synthesis is also discussed.