Journal articles: 'RNA Syntheis'

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ZACHLEDER, V., and I. ŠETLÍK. "Distinct controls of DNA replication and of nuclear division in the cell cycles of the chlorococcal alga Scenedesmus quadricauda." Journal of Cell Science 91, no. 4 (December 1, 1988): 531–39. http://dx.doi.org/10.1242/jcs.91.4.531.

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In the course of the cell cycle of Scenedesmus quadricauda, the syntheses of RNA and total protein occur in steps. Each step represents an approximate doubling of the preceding amount of RNA or protein per cell. The increase in protein content per cell runs parallel to, but with a constant delay behind, the corresponding RNA steps. When protein synthesis is suppressed (e.g. by maintaining the cells in the dark) after an RNA synthesis step has already occurred the cells double their DNA content, but no corresponding nuclear division occurs and uninuclear daughter cells with double the amount of DNA may be formed. Under conditions of phosphorus or nitrogen starvation RNA synthesis is stopped while protein synthesis continues. In this case, the number of DNA replication rounds corresponds to the reduced RNA content while the number of nuclear divisions tends to follow the number of protein synthesis steps until one genome per nucleus is attained. These results indicate that with each doubling of RNA content the cells become committed to DNA replication, while doubling of protein content is required for the commitment to the corresponding nuclear divisions.

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Macnaughton, Thomas B., Stephanie T. Shi, Lucy E. Modahl, and Michael M. C. Lai. "Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases." Journal of Virology 76, no. 8 (April 15, 2002): 3920–27. http://dx.doi.org/10.1128/jvi.76.8.3920-3927.2002.

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ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

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Harold, Cecelia M., Amber F. Buhagiar, Yan Cheng, and Susan J. Baserga. "Ribosomal RNA Transcription Regulation in Breast Cancer." Genes 12, no. 4 (March 29, 2021): 502. http://dx.doi.org/10.3390/genes12040502.

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Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar structure is indicative of poor cancer prognoses. In recent years, it has been shown that ribosome biogenesis, and rDNA transcription in particular, is dysregulated in cancer cells. Coupled with advancements in screening technology that allowed for the discovery of novel drugs targeting RNA Polymerase I, this transcriptional machinery is an increasingly viable target for cancer therapies. In this review, we discuss ribosome biogenesis in breast cancer and the different cellular pathways involved. Moreover, we discuss current therapeutics that have been found to affect rDNA transcription and more novel drugs that target rDNA transcription machinery as a promising avenue for breast cancer treatment.

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Harrington, C. A., and D. M. Chikaraishi. "Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene." Molecular and Cellular Biology 7, no. 1 (January 1987): 314–25. http://dx.doi.org/10.1128/mcb.7.1.314-325.1987.

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The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

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Harrington, C. A., and D. M. Chikaraishi. "Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene." Molecular and Cellular Biology 7, no. 1 (January 1987): 314–25. http://dx.doi.org/10.1128/mcb.7.1.314.

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The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

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Tian, Q., G. S. Kopf, R. S. Brown, and H. Tseng. "Function of basonuclin in increasing transcription of the ribosomal RNA genes during mouse oogenesis." Development 128, no. 3 (February 1, 2001): 407–16. http://dx.doi.org/10.1242/dev.128.3.407.

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Active protein synthesis during early oogenesis requires accelerated transcription of ribosomal RNA genes (rDNAs). In response to this demand, rDNAs are amplified more than 1000-fold early in Xenopus oogenesis. Here, we report evidence that rDNA is not amplified in mouse oocytes, but these cells may instead employ the zinc-finger protein basonuclin, a putative rDNA transcription factor, to enhance rRNA synthesis. This conclusion is based on observations that basonuclin is localized in the nucleolus in the mouse oocyte early in its growth phase, when rRNA transcription is highly active; and that the binding sites of basonuclin zinc fingers on the human and mouse rDNA promoters are homologous. In a co-transfection assay, basonuclin can elevate transcription from an rDNA promoter, and its zinc-finger domain can inhibit RNA polymerase I transcription, as detected by a run-on assay, in growing mouse oocytes.

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Bednarska, Elżbieta. "Ultrastructural and metabolic transformations of differentiating Hyacinthus orientalis L, pollen grain cells. I. RNA and protein synthesis." Acta Societatis Botanicorum Poloniae 53, no. 2 (2014): 145–58. http://dx.doi.org/10.5586/asbp.1984.014.

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RNA and protein synthesis were investigated in generative and vegetative cells during maturation of pollen grains. The rate of RNA and protein synthesis was analysed in reference to the successive interphase periods of the life cycle of pollen cells as well as against the background of the growth dynamics of the cell volume. The results of studies demonstrated that the pollen grain increases in size owing to the growth of the vegetative cell. The generative one does not grow. RNA synthesis and that of proteins in differentiating pollen cells has a different course. In the growing vegetative cell it lasts longer and is more intensive than in the generative cell which does not grow. RNA and protein synthesis in the vegetative cell take place in the period from the callose stage to the stage of lemon-shaped generative cell, that is in the period of phases G1, S and G2. This synthesis is positively correlated with the growth of the pollen grain. RNA and protein synthesis in the generative cell comprises the period from the callose-less lenticular stage to the stage of spherical generative cell, that is the phases S and early phase G2. These results suggest that in the vegetative cell RNA and protein synthesis is utilised above all to increase of its cell, instead in non growing generative cell protein synthese is probably limited mostly to a histones and enzymatic proteins serving for the DNA replication process.

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Sivakumaran, K., and C. Cheng Kao. "Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase." Journal of Virology 73, no. 8 (August 1, 1999): 6415–23. http://dx.doi.org/10.1128/jvi.73.8.6415-6423.1999.

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ABSTRACT In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3′ end of a template requires one nucleotide 3′ of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3′ end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the −1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5′ of the initiation site.

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Beerens, Nancy, Barbara Selisko, Stefano Ricagno, Isabelle Imbert, Linda van der Zanden, Eric J. Snijder, and Bruno Canard. "De Novo Initiation of RNA Synthesis by the Arterivirus RNA-Dependent RNA Polymerase." Journal of Virology 81, no. 16 (May 30, 2007): 8384–95. http://dx.doi.org/10.1128/jvi.00564-07.

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ABSTRACT All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3′ end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3′-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

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Izumikawa, Keiichi, Hideaki Ishikawa, Harunori Yoshikawa, Sally Fujiyama, Akira Watanabe, Hiroyuki Aburatani, Hiroyuki Tachikawa, et al. "LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA." Nucleic Acids Research 47, no. 19 (September 2, 2019): 10357–72. http://dx.doi.org/10.1093/nar/gkz747.

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Abstract Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine–binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

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Fahrenbach, Albert C. "Template-directed nonenzymatic oligonucleotide synthesis: lessons from synthetic chemistry." Pure and Applied Chemistry 87, no. 2 (February 1, 2015): 205–18. http://dx.doi.org/10.1515/pac-2014-1004.

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AbstractThe nonenzymatic synthesis of nucleic acids, in particular, RNA, and the template-directed synthesis of artificial organic molecules, such as macrocycles, catenanes and rotaxanes, have both undergone significant development since the last half of the 20th century. The intersection of these two fields affords insights into how template effects can lead to information copying and storage at the molecular level. Mechanistic examples of model template-directed RNA replication experiments as well as those for totally artificial organic template-directed syntheses will be discussed. The fact that templates typically bind to their reacted products more tightly than their unreacted substrates may be a mechanistic feature necessary to store information in the form of nucleic acids. Understanding the mechanisms of nonenzymatic RNA synthesis is not only essential for testing the RNA world hypothesis in the context of the origin of life on Earth and other planetary bodies, but may one day afford chemists the insights to construct their own artificial molecular replicators.

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Modahl, Lucy E., Thomas B. Macnaughton, Nongliao Zhu, Deborah L. Johnson, and Michael M. C. Lai. "RNA-Dependent Replication and Transcription of Hepatitis Delta Virus RNA Involve Distinct Cellular RNA Polymerases." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 6030–39. http://dx.doi.org/10.1128/mcb.20.16.6030-6039.2000.

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ABSTRACT Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of α-amanitin (<3 μg/ml) and could be partially restored by an α-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by α-amanitin to a concentration higher than 25 μg/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

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García-Martínez, José, Daniel A. Medina, Pablo Bellvís, Mai Sun, Patrick Cramer, Sebastián Chávez, and José E. Pérez-Ortín. "The total mRNA concentration buffering system in yeast is global rather than gene-specific." RNA 27, no. 10 (July 16, 2021): 1281–90. http://dx.doi.org/10.1261/rna.078774.121.

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Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentrations and their synthesis rates in nonsense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one.

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Lai, Vicky C. H., C. Cheng Kao, Eric Ferrari, Justin Park, Annette S. Uss, Jacquelyn Wright-Minogue, Zhi Hong, and Johnson Y. N. Lau. "Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase." Journal of Virology 73, no. 12 (December 1, 1999): 10129–36. http://dx.doi.org/10.1128/jvi.73.12.10129-10136.1999.

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ABSTRACT Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BΔCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (ΔCT179 and ΔCT218) that lacked RdRp activities.

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Mairhofer, Elisabeth, Elisabeth Fuchs, and Ronald Micura. "Facile synthesis of a 3-deazaadenosine phosphoramidite for RNA solid-phase synthesis." Beilstein Journal of Organic Chemistry 12 (November 28, 2016): 2556–62. http://dx.doi.org/10.3762/bjoc.12.250.

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Access to 3-deazaadenosine (c3A) building blocks for RNA solid-phase synthesis represents a severe bottleneck in modern RNA research, in particular for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report the 5-step synthesis of a c3A phosphoramidite from cost-affordable starting materials. The key reaction is a silyl-Hilbert–Johnson nucleosidation using unprotected 6-amino-3-deazapurine and benzoyl-protected 1-O-acetylribose. The novel path is superior to previously described syntheses in terms of efficacy and ease of laboratory handling.

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Röthlisberger, Pascal, Christian Berk, and Jonathan Hall. "RNA Chemistry for RNA Biology." CHIMIA International Journal for Chemistry 73, no. 5 (May 29, 2019): 368–73. http://dx.doi.org/10.2533/chimia.2019.368.

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Advances in the chemical synthesis of RNA have opened new possibilities to address current questions in RNA biology. Access to site-specifically modified oligoribonucleotides is often a pre-requisite for RNA chemical-biology projects. Driven by the enormous research efforts for development of oligonucleotide therapeutics, a wide range of chemical modifications have been developed to modulate the intrinsic properties of nucleic acids in order to fit their use as therapeutics or research tools. The RNA synthesis platform, supported by the NCCR RNA & Disease, aims to provide access to a large variety of chemically modified nucleic acids. In this review, we describe some of the recent projects that involved work of the platform and highlight how RNA chemistry supports new discoveries in RNA biology.

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Zhong, Weidong, Haoyun An, Dinesh Barawkar, and Zhi Hong. "Dinucleotide Analogues as Novel Inhibitors of RNA-Dependent RNA Polymerase of Hepatitis C Virus." Antimicrobial Agents and Chemotherapy 47, no. 8 (August 2003): 2674–81. http://dx.doi.org/10.1128/aac.47.8.2674-2681.2003.

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ABSTRACT Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5′-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.

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Grewal, Savraj S., Justin R. Evans, and Bruce A. Edgar. "Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway." Journal of Cell Biology 179, no. 6 (December 17, 2007): 1105–13. http://dx.doi.org/10.1083/jcb.200709044.

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Synthesis of ribosomal RNA (rRNA) is a key step in ribosome biogenesis and is essential for cell growth. Few studies, however, have investigated rRNA synthesis regulation in vivo in multicellular organisms. Here, we present a genetic analysis of transcription initiation factor IA (TIF-IA), a conserved RNA polymerase I transcription factor. Drosophila melanogaster Tif-IA−/− mutants have reduced levels of rRNA synthesis and sustain a developmental arrest caused by a block in cellular growth. We find that the target of rapamycin (TOR) pathway regulates TIF-IA recruitment to rDNA. Furthermore, we show that the TOR pathway regulates rRNA synthesis in vivo and that TIF-IA overexpression can maintain rRNA transcription when TOR activity is reduced in developing larvae. We propose that TIF-IA acts in vivo as a downstream growth–regulatory target of the TOR pathway. Overexpression of TIF-IA also elevates levels of both 5S RNA and messenger RNAs encoding ribosomal proteins. Stimulation of rRNA synthesis by TIF-IA may therefore provide a feed-forward mechanism to coregulate the levels of other ribosome components.

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Engel, Krysta L., Sarah L. French, Olga V. Viktorovskaya, Ann L. Beyer, and David A. Schneider. "Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I." Molecular and Cellular Biology 35, no. 13 (April 27, 2015): 2321–31. http://dx.doi.org/10.1128/mcb.01499-14.

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Spt6 (suppressor ofTy6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele,spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression ofRRN3rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

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Ren, M., A. Villamarin, A. Shih, E. Coutavas, M. S. Moore, M. LoCurcio, V. Clarke, J. D. Oppenheim, P. D'Eustachio, and M. G. Rush. "Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing." Molecular and Cellular Biology 15, no. 4 (April 1995): 2117–24. http://dx.doi.org/10.1128/mcb.15.4.2117.

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The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

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Wang, Lei, Neil A. Smith, Lan Zhang, Elizabeth S. Dennis, Peter M. Waterhouse, Peter J. Unrau, and Ming-Bo Wang. "Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer." Functional Plant Biology 35, no. 11 (2008): 1091. http://dx.doi.org/10.1071/fp08118.

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RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract.

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Ranjith-Kumar, C. T., Les Gutshall, Min-Ju Kim, Robert T. Sarisky, and C. Cheng Kao. "Requirements for De Novo Initiation of RNA Synthesis by Recombinant Flaviviral RNA-Dependent RNA Polymerases." Journal of Virology 76, no. 24 (December 15, 2002): 12526–36. http://dx.doi.org/10.1128/jvi.76.24.12526-12536.2002.

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ABSTRACT RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg2+ and Mn2+ on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn2+ was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn2+. These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.

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Sivakumaran, K., M. Hema, and C. Cheng Kao. "Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts." Journal of Virology 77, no. 10 (May 15, 2003): 5703–11. http://dx.doi.org/10.1128/jvi.77.10.5703-5711.2003.

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ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

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Vallett, S. M., M. Brudnak, M. Pellegrini, and H. W. Weber. "In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum." Molecular and Cellular Biology 13, no. 2 (February 1993): 928–33. http://dx.doi.org/10.1128/mcb.13.2.928-933.1993.

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The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

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Vallett, S. M., M. Brudnak, M. Pellegrini, and H. W. Weber. "In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum." Molecular and Cellular Biology 13, no. 2 (February 1993): 928–33. http://dx.doi.org/10.1128/mcb.13.2.928.

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The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

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Rohayem, Jacques, Katrin Jäger, Ivonne Robel, Ulrike Scheffler, Achim Temme, and Wolfram Rudolph. "Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis." Journal of General Virology 87, no. 9 (September 1, 2006): 2621–30. http://dx.doi.org/10.1099/vir.0.81802-0.

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Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

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Kao, C. Cheng, Xueyong Yang, Allen Kline, Q. May Wang, Donna Barket, and Beverly A. Heinz. "Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase." Journal of Virology 74, no. 23 (December 1, 2000): 11121–28. http://dx.doi.org/10.1128/jvi.74.23.11121-11128.2000.

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ABSTRACT The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

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van Dijk, Alberdina A., Eugene V. Makeyev, and Dennis H. Bamford. "Initiation of viral RNA-dependent RNA polymerization." Journal of General Virology 85, no. 5 (May 1, 2004): 1077–93. http://dx.doi.org/10.1099/vir.0.19731-0.

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This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3′ terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.

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Bellinder, Robin R., Kriton K. Hatzios, and Henry P. Wilson. "Mode of Action Investigations with the Herbicides HOE-39866 and SC-0224." Weed Science 33, no. 6 (November 1985): 779–85. http://dx.doi.org/10.1017/s004317450008334x.

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In laboratory experiments, the effects of the herbicides HOE-39866 [ammonium (3-amino-3-carboxypropyl)-methylphosphinate] and SC-0224 (trimethylsulfonium carboxymethylaminomethylphosphonate) on the incorporation of NaH14CO3, [14C]-leucine, [14C]-uracil, and [14C]-acetate into enzymatically isolated soybean [Glycine max(L.) Merr. ‘Essex’] cells were evaluated to assess the activity of these herbicides on CO2fixation, protein, ribonucleic acid (RNA), and lipid syntheses. At low concentrations neither compound exhibited rapid or distinct inhibitions of any process as might be expected in the case of inhibition of a primary target site. Photosynthesis was the process least affected. At equimolar concentrations, protein and RNA syntheses were more sensitive to HOE-39866 than to SC-0224 while the reverse occurred in lipid synthesis. Protein synthesis appears to be a possible target site that may be involved in the herbicidal action of these two compounds.

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Albert, Benjamin, Jorge Perez-Fernandez, Isabelle Léger-Silvestre, and Olivier Gadal. "Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?" Genetics Research International 2012 (January 12, 2012): 1–13. http://dx.doi.org/10.1155/2012/276948.

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Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

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VANZI, F. "Protein synthesis by single ribosomes." RNA 9, no. 10 (October 1, 2003): 1174–79. http://dx.doi.org/10.1261/rna.5800303.

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Becker, Sidney, Jonas Feldmann, Stefan Wiedemann, Hidenori Okamura, Christina Schneider, Katharina Iwan, Antony Crisp, Martin Rossa, Tynchtyk Amatov, and Thomas Carell. "Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides." Science 366, no. 6461 (October 3, 2019): 76–82. http://dx.doi.org/10.1126/science.aax2747.

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Theories about the origin of life require chemical pathways that allow formation of life’s key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5′-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.

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Girke, Philipp, and Wolfgang Seufert. "Compositional reorganization of the nucleolus in budding yeast mitosis." Molecular Biology of the Cell 30, no. 5 (March 2019): 591–606. http://dx.doi.org/10.1091/mbc.e18-08-0524.

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The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.

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von Walden, Ferdinand. "Ribosome biogenesis in skeletal muscle: coordination of transcription and translation." Journal of Applied Physiology 127, no. 2 (August 1, 2019): 591–98. http://dx.doi.org/10.1152/japplphysiol.00963.2018.

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Skeletal muscle mass responds in a remarkable manner to alterations in loading and use. It has long been clear that skeletal muscle hypertrophy can be prevented by inhibiting RNA synthesis. Since 80% of the cell’s total RNA has been estimated to be rRNA, this finding indicates that de novo production of rRNA via transcription of the corresponding genes is important for such hypertrophy to occur. Transcription of rDNA by RNA Pol I is the rate-limiting step in ribosome biogenesis, indicating in turn that this biogenesis strongly influences the hypertrophic response. The present minireview focuses on 1) a brief description of the key steps in ribosome biogenesis and the relationship of this process to skeletal muscle mass and 2) the coordination of ribosome biogenesis and protein synthesis for growth or atrophy, as exemplified by the intracellular AMPK and mTOR pathways.

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Sorino, Cristina, Valeria Catena, Tiziana Bruno, Francesca De Nicola, Stefano Scalera, Gianluca Bossi, Francesca Fabretti, et al. "Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription." Nucleic Acids Research 48, no. 11 (May 18, 2020): 5891–906. http://dx.doi.org/10.1093/nar/gkaa344.

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Abstract Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

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Cazenave, C., and O. C. Uhlenbeck. "RNA template-directed RNA synthesis by T7 RNA polymerase." Proceedings of the National Academy of Sciences 91, no. 15 (July 19, 1994): 6972–76. http://dx.doi.org/10.1073/pnas.91.15.6972.

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37

Cao, Dongdong, Yunrong Gao, and Bo Liang. "Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes." Viruses 13, no. 5 (May 5, 2021): 834. http://dx.doi.org/10.3390/v13050834.

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RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

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38

Kim, Min-Ju, Weidong Zhong, Zhi Hong, and C. Cheng Kao. "Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase." Journal of Virology 74, no. 22 (November 15, 2000): 10312–22. http://dx.doi.org/10.1128/jvi.74.22.10312-10322.2000.

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ABSTRACT The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3′ end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1–7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2′-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2′-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2′-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.

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Resa-Infante, Patricia, Núria Jorba, Rocio Coloma, and Juan Ortin. "The influenza virus RNA synthesis machine." RNA Biology 8, no. 2 (March 2011): 207–15. http://dx.doi.org/10.4161/rna.8.2.14513.

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NATHANSON, L. "Nuclear protein synthesis: A re-evaluation." RNA 9, no. 1 (January 1, 2003): 9–13. http://dx.doi.org/10.1261/rna.2990203.

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41

Luo, Guangxiang, Robert K. Hamatake, Danielle M. Mathis, Jason Racela, Karen L. Rigat, Julie Lemm, and Richard J. Colonno. "De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus." Journal of Virology 74, no. 2 (January 15, 2000): 851–63. http://dx.doi.org/10.1128/jvi.74.2.851-863.2000.

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ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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Guo, Bin, Devasier Bennet, Daniel J. Belcher, Hyo-Gun Kim, and Gustavo A. Nader. "Chemotherapy agents reduce protein synthesis and ribosomal capacity in myotubes independent of oxidative stress." American Journal of Physiology-Cell Physiology 321, no. 6 (December 1, 2021): C1000—C1009. http://dx.doi.org/10.1152/ajpcell.00116.2021.

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Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side effects associated with CAs promote muscle loss by incompletely understood mechanisms. To address this problem, we first identified how oxidative stress impairs protein synthesis in C2C12 myotubes. Transient elevations in reactive oxygen species (ROS) resulted in protein synthesis deficits and reduced ribosomal (r)RNA levels. Oxidative stress did not reduce rRNA gene (rDNA) transcription, but it caused an increase in rRNA and protein oxidation. To determine whether CAs affect protein synthesis independent of oxidative stress, we exposed myotubes to Paclitaxel (PTX), Doxorubicin (DXR), or Marizomib (Mzb) at doses that did result in elevated ROS levels (sub-ROS). Exposure to CAs reduced protein synthesis and rRNA levels, but unlike oxidative stress, sub-ROS exposures impaired rDNA transcription. These results indicate that although oxidative stress disrupts protein synthesis by compromising ribosomal quantity and quality, CAs at sub-ROS doses compromise protein synthesis and ribosomal capacity, at least in part, by reducing rDNA transcription. Therefore, CAs negatively impact protein synthesis by causing oxidative stress in addition to directly reducing the ribosomal capacity of myotubes in a ROS-independent manner.

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Guo, Hui, Mengyue Fan, Zengjin Li, Wei Tang, and Xinrui Duan. "Ratiometric RNA aptamer/fluorophore complex for RNA synthesis detection." Analytical Methods 10, no. 47 (2018): 5629–33. http://dx.doi.org/10.1039/c8ay01880d.

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Pérez-Ortín, José E., Adriana Mena, Marina Barba-Aliaga, Abhyudai Singh, Sebastián Chávez, and José García-Martínez. "Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast." PLOS Genetics 17, no. 4 (April 7, 2021): e1009520. http://dx.doi.org/10.1371/journal.pgen.1009520.

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The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

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Steil, Benjamin P., and David J. Barton. "Poliovirus cis-Acting Replication Element-Dependent VPg Uridylylation Lowers the Km of the Initiating Nucleoside Triphosphate for Viral RNA Replication." Journal of Virology 82, no. 19 (July 23, 2008): 9400–9408. http://dx.doi.org/10.1128/jvi.00427-08.

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ABSTRACT Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5′ terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5′ end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpUOH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpUOH-independent manner, CRE-dependent VPgpUpUOH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the Km of UTP required for viral RNA replication and that CRE-dependent VPgpUpUOH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.

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Ahmed, Yasar Luqman, and Ralf Ficner. "RNA synthesis and purification for structural studies." RNA Biology 11, no. 5 (February 10, 2014): 427–32. http://dx.doi.org/10.4161/rna.28076.

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Puthenvedu, Deepa, Teresa Janas, Irene Majerfeld, Mali Illangasekare, and Michael Yarus. "Poly(U) RNA-templated synthesis of AppA." RNA 21, no. 10 (August 13, 2015): 1818–25. http://dx.doi.org/10.1261/rna.052696.115.

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Robert, F. "Chlorolissoclimides: New inhibitors of eukaryotic protein synthesis." RNA 12, no. 5 (March 15, 2006): 717–25. http://dx.doi.org/10.1261/rna.2346806.

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Feng, Shuang, and James L. Manley. "Beyond rRNA: nucleolar transcription generates a complex network of RNAs with multiple roles in maintaining cellular homeostasis." Genes & Development 36, no. 15-16 (August 1, 2022): 876–86. http://dx.doi.org/10.1101/gad.349969.122.

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Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes ∼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.

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Takeda, Yoshihiko, Patricia Caudell, Giuia Grady, Grace Wang, Akira Suwa, Gordon C. Sharp, William S. Dynan, and John A. Hardin. "Human RNA Helicase A Is a Lupus Autoantigen That Is Cleaved During Apoptosis." Journal of Immunology 163, no. 11 (December 1, 1999): 6269–74. http://dx.doi.org/10.4049/jimmunol.163.11.6269.

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Abstract:

Abstract Proteolytic cleavage by caspases is the central event in cells undergoing apoptosis. Cleaved proteins are often targeted by autoantibodies, suggesting that the cleavage of self Ags enhances immunogenicity and is prone to induce an autoimmune response. We found autoantibodies that immunoprecipitated a 140-kDa RNA-associated protein, provisionally designated Pa, in 11 of 350 patient sera that were positive for antinuclear Abs in an immunofluorescence test. The Pa protein gave rise to three fragments with m.w. ranging from 120–130 kDa during anti-Fas-activated apoptosis. Pure caspase-3 cleaved the Pa protein into a 130-kDa fragment corresponding to the largest of these three products. Peptide sequence analysis of a tryptic digest from immunoaffinity-purified Pa showed 100% identity to human RNA helicase A (RHA). The identity of Pa with RHA was further confirmed by immunoblotting with rabbit anti-RHA Ab using anti-Pa immunoprecipitates as substrates. All 10 anti-RHA-positive patients who were clinically analyzed were diagnosed as having systemic lupus erythematosus, and 7 of them had lupus nephritis. RHA is a multifunctional protein with roles in cellular RNA synthesis and processing. Inactivation of RHA by cleavage may be an important part of the process leading to programmed cell death. The cleaved RHA fragments that are produced during apoptosis may trigger an autoimmune response in systemic lupus erythematosus.

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Journal articles on the topic 'RNA Syntheis'

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