Journal articles: 'Multimeric RNA'

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Lee, Ji-Hye, Yeon Bin Chung, Jong Hyeon Seok, Kang Rok Han, Sella Kim, and Kyung Hyun Kim. "Structural basis for VPg-induced formation of RNA-dependent RNA polymerase multimeric complexes." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1601. http://dx.doi.org/10.1107/s2053273314083983.

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Norovirus is the leading cause of epidemic acute, nonbacterial gastroenteritis, and adopts de novo and VPg (Virion protein genome linked)-primed RNA synthesis by RNA-dependent RNA polymerase (RdRp). To understand the interaction between RdRp and VPg in replication of murine norovirus-1 (MNV-1), we determined the crystal structure of MNV-1 RdRp-VPg(1-73)-RNA complex. VPg was bound to the base of the palm domain and the tip of the fingers domain of RdRp simultaneously, but RNA template could not be modeled. The binding affinity constants (Kd) for RdRp-VPg was 3.7411.57 nM and VPg(1-73) showed approximately 90-fold less affinity than that of full-length VPg. In addition to this multiple binding mode, VPg enhanced the interactions of RdRp hexamers, leading to the formation of high-order multimers or tubular fibrils with significantly increased polymerase activity, confirmed by electron microscopic and biochemical studies. Our data indicated that MNV-1 VPg with helical structure was bound to RdRp at multiple sites and induces RdRp multimerization in viral replication. The multimers of RdRp-VPg-RNA can provide a mechanistic understanding of viral polymerase multimeric arrays and a new tool for development of antivirals to control norovirus outbreaks. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs (A085119 K.H.K), Basic Science Research Program through the National Research Foundation (NRF-2013R1A1A2064940, L.J-H), Korea University Grant (L.J-H), and the BK21 plus program of the Ministry of Education, Korea.

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Guo, Li-Hua, Yun-He Cao, Da-Wei Li, Sheng-Niao Niu, Zhu-Nan Cai, Cheng-Gui Han, Ya-Feng Zhai, and Jia-Lin Yu. "Analysis of Nucleotide Sequences and Multimeric Forms of a Novel Satellite RNA Associated with Beet Black Scorch Virus." Journal of Virology 79, no. 6 (March 15, 2005): 3664–74. http://dx.doi.org/10.1128/jvi.79.6.3664-3674.2005.

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ABSTRACT The full-length sequence of a satellite RNA (sat-RNA) of Beet black scorch virus isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to Chenopodium amaranticolor with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5- or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5′ or the 3′ termini. Therefore, sequences at both the 5′ and the 3′ ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5′ end sequences and/or by internal initiation at the 3′ ends of multimeric junctions.

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Lin, Brian C., Dawn A. Defenbaugh, and John L. Casey. "Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity." Journal of Virology 84, no. 3 (November 18, 2009): 1406–13. http://dx.doi.org/10.1128/jvi.01723-09.

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ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

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CLERTE, C. "Characterization of multimeric complexes formed by the human PTB1 protein on RNA." RNA 12, no. 3 (March 1, 2006): 457–75. http://dx.doi.org/10.1261/rna.2178406.

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Kim, Dajeong, Sangwoo Han, Yoonbin Ji, Sunghyun Moon, Hyangsu Nam, and Jong Bum Lee. "Multimeric RNAs for efficient RNA-based therapeutics and vaccines." Journal of Controlled Release 345 (May 2022): 770–85. http://dx.doi.org/10.1016/j.jconrel.2022.03.052.

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Heym, Roland G., Dennis Zimmermann, Franziska T. Edelmann, Lars Israel, Zeynep Ökten, David R. Kovar, and Dierk Niessing. "In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation." Journal of Cell Biology 203, no. 6 (December 23, 2013): 971–84. http://dx.doi.org/10.1083/jcb.201302095.

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The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

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Seyhan, Attila A., Alexander V. Vlassov, and Brian H. Johnston. "RNA Interference from Multimeric shRNAs Generated by Rolling Circle Transcription." Oligonucleotides 16, no. 4 (December 2006): 353–63. http://dx.doi.org/10.1089/oli.2006.16.353.

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8

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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Sharon, G., T. J. Burkett, and D. J. Garfinkel. "Efficient homologous recombination of Ty1 element cDNA when integration is blocked." Molecular and Cellular Biology 14, no. 10 (October 1994): 6540–51. http://dx.doi.org/10.1128/mcb.14.10.6540-6551.1994.

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Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.

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Sharon, G., T. J. Burkett, and D. J. Garfinkel. "Efficient homologous recombination of Ty1 element cDNA when integration is blocked." Molecular and Cellular Biology 14, no. 10 (October 1994): 6540–51. http://dx.doi.org/10.1128/mcb.14.10.6540.

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Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.

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Dalmay, T., and L. Rubino. "The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA." Archives of Virology 138, no. 1-2 (March 1994): 161–67. http://dx.doi.org/10.1007/bf01310047.

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12

Olotu, Fisayo A., Kehinde F. Omolabi, and Mahmoud E. S. Soliman. "Piece of the puzzle: Remdesivir disassembles the multimeric SARS-CoV-2 RNA-dependent RNA polymerase complex." Cell Biochemistry and Biophysics 79, no. 2 (April 1, 2021): 175–87. http://dx.doi.org/10.1007/s12013-021-00977-y.

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Rech, Bruna, and Fernando A. Gonzales-Zubiate. "Mechanisms of Nuclear Transport in the cell: RNA exosome in Saccharomyces cerevisiae." Bionatura 5, no. 4 (November 15, 2020): 1423–26. http://dx.doi.org/10.21931/rb/2020.05.04.25.

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Ribonucleases (RNases) functions in the cell include precise maturation of non- coding RNAs and degradation of specific RNA transcripts that are no longer necessary. RNAses are present in the cell as single units or assembled as multimeric complexes; one of these complexes is the RNA exosome, a highly conserved complex essential for RNA processing and degradation. In the yeast Saccharomyces cerevisiae, the RNA exosome comprises eleven subunits, two with catalytic activity: Rrp6 and Rrp44, where the Rrp6 subunit is exclusively nuclear. Despite the RNA exosome has been intensively investigated since its discovery in 1997, only a few studies were accomplished concerning its nuclear transport. This review describes recent research about cellular localization and transport of this essential complex.

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Lange, Heike, and Dominique Gagliardi. "Catalytic activities, molecular connections, and biological functions of plant RNA exosome complexes." Plant Cell 34, no. 3 (December 25, 2021): 967–88. http://dx.doi.org/10.1093/plcell/koab310.

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Abstract RNA exosome complexes provide the main 3′–5′-exoribonuclease activities in eukaryotic cells and contribute to the maturation and degradation of virtually all types of RNA. RNA exosomes consist of a conserved core complex that associates with exoribonucleases and with multimeric cofactors that recruit the enzyme to its RNA targets. Despite an overall high level of structural and functional conservation, the enzymatic activities and compositions of exosome complexes and their cofactor modules differ among eukaryotes. This review highlights unique features of plant exosome complexes, such as the phosphorolytic activity of the core complex, and discusses the exosome cofactors that operate in plants and are dedicated to the maturation of ribosomal RNA, the elimination of spurious, misprocessed, and superfluous transcripts, or the removal of mRNAs cleaved by the RNA-induced silencing complex and other mRNAs prone to undergo silencing.

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Bryk, Mary, Mukti Banerjee, Darryl Conte, and M. Joan Curcio. "The Sgs1 Helicase of Saccharomyces cerevisiae Inhibits Retrotransposition of Ty1 Multimeric Arrays." Molecular and Cellular Biology 21, no. 16 (August 15, 2001): 5374–88. http://dx.doi.org/10.1128/mcb.21.16.5374-5388.2001.

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ABSTRACT Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32- to 79-fold in sgs1Δ mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1Δmutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1Δ mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.

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Jiang, X., and J. G. Belasco. "Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA." Proceedings of the National Academy of Sciences 101, no. 25 (June 14, 2004): 9211–16. http://dx.doi.org/10.1073/pnas.0401382101.

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Molina-Serrano, Diego, Loreto Suay, María L. Salvador, Ricardo Flores, and José-Antonio Daròs. "Processing of RNAs of the Family Avsunviroidae in Chlamydomonas reinhardtii Chloroplasts." Journal of Virology 81, no. 8 (February 7, 2007): 4363–66. http://dx.doi.org/10.1128/jvi.02556-06.

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ABSTRACT The family Avsunviroidae comprises four viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus- and minus-strand dimeric transcripts of representative members of the family Avsunviroidae. Despite the absence of viroid RNA-RNA transcription, the C. reinhardtii-based system can be used to address intriguing questions about viroid RNA processing and, in particular, about the cellular factors involved in cleavage and ligation.

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Buzayan, Jamal M., Wayne L. Gerlach, George Bruening, Paul Keese, and Allan R. Gould. "Nucleotide sequence of satellite tobacco ringspot virus RNA and its relationship to multimeric forms." Virology 151, no. 2 (June 1986): 186–99. http://dx.doi.org/10.1016/0042-6822(86)90041-3.

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Levanova, Alesia A., Mirka Lampi, Kiira Kalke, Veijo Hukkanen, Minna M. Poranen, and Katri Eskelin. "Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation." Pharmaceuticals 15, no. 2 (February 21, 2022): 261. http://dx.doi.org/10.3390/ph15020261.

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RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.

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Robbins, Susan G., Mark F. Frana, John J. McGowan, John F. Boyle, and Kathryn V. Holmest. "RNA-binding proteins of coronavirus MHV: Detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay." Virology 150, no. 2 (April 1986): 402–10. http://dx.doi.org/10.1016/0042-6822(86)90305-3.

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Matzat, Leah H., Stephen Berberoglu, and Lyne Lévesque. "Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins." Molecular Biology of the Cell 19, no. 1 (January 2008): 327–38. http://dx.doi.org/10.1091/mbc.e07-03-0255.

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Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

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Opi, Sandrine, Hiroaki Takeuchi, Sandra Kao, Mohammad A. Khan, Eri Miyagi, Ritu Goila-Gaur, Yasumasa Iwatani, Judith G. Levin, and Klaus Strebel. "Monomeric APOBEC3G Is Catalytically Active and Has Antiviral Activity." Journal of Virology 80, no. 10 (May 15, 2006): 4673–82. http://dx.doi.org/10.1128/jvi.80.10.4673-4682.2006.

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ABSTRACT APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.

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Ferbeyre, Gerardo, James M. Smith, and Robert Cedergren. "Schistosome Satellite DNA Encodes Active Hammerhead Ribozymes." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3880–88. http://dx.doi.org/10.1128/mcb.18.7.3880.

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ABSTRACT Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smα repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in bothcis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smα ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum orHeterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

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Chen, Xiaojiang S. "Insights into the Structures and Multimeric Status of APOBEC Proteins Involved in Viral Restriction and Other Cellular Functions." Viruses 13, no. 3 (March 17, 2021): 497. http://dx.doi.org/10.3390/v13030497.

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Apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) proteins belong to a family of deaminase proteins that can catalyze the deamination of cytosine to uracil on single-stranded DNA or/and RNA. APOBEC proteins are involved in diverse biological functions, including adaptive and innate immunity, which are critical for restricting viral infection and endogenous retroelements. Dysregulation of their functions can cause undesired genomic mutations and RNA modification, leading to various associated diseases, such as hyper-IgM syndrome and cancer. This review focuses on the structural and biochemical data on the multimerization status of individual APOBECs and the associated functional implications. Many APOBECs form various multimeric complexes, and multimerization is an important way to regulate functions for some of these proteins at several levels, such as deaminase activity, protein stability, subcellular localization, protein storage and activation, virion packaging, and antiviral activity. The multimerization of some APOBECs is more complicated than others, due to the associated complex RNA binding modes.

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de Jong, Annika, Richard J. Dirven, Johan Boender, Ferdows Atiq, Seyed Yahya Anvar, Frank W. G. Leebeek, Bart J. M. van Vlijmen, and Jeroen Eikenboom. "Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA." Thrombosis and Haemostasis 120, no. 11 (August 15, 2020): 1569–79. http://dx.doi.org/10.1055/s-0040-1715442.

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AbstractVon Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

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Buxton, Penelope, Gilda Tachedjian, and Johnson Mak. "Analysis of the Contribution of Reverse Transcriptase and Integrase Proteins to Retroviral RNA Dimer Conformation." Journal of Virology 79, no. 10 (May 15, 2005): 6338–48. http://dx.doi.org/10.1128/jvi.79.10.6338-6348.2005.

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ABSTRACT All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

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Song, Sang Ik, and W. Allen Miller. "cis and trans Requirements for Rolling Circle Replication of a Satellite RNA." Journal of Virology 78, no. 6 (March 15, 2004): 3072–82. http://dx.doi.org/10.1128/jvi.78.6.3072-3082.2004.

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ABSTRACT Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.

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Moradian, Hanieh, Andreas Lendlein, and Manfred Gossen. "Strategies for simultaneous and successive delivery of RNA." Journal of Molecular Medicine 98, no. 12 (November 4, 2020): 1767–79. http://dx.doi.org/10.1007/s00109-020-01956-1.

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AbstractAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

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Lee, Benjamin D., and Eugene V. Koonin. "Viroids and Viroid-like Circular RNAs: Do They Descend from Primordial Replicators?" Life 12, no. 1 (January 12, 2022): 103. http://dx.doi.org/10.3390/life12010103.

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Viroids are a unique class of plant pathogens that consist of small circular RNA molecules, between 220 and 450 nucleotides in size. Viroids encode no proteins and are the smallest known infectious agents. Viroids replicate via the rolling circle mechanism, producing multimeric intermediates which are cleaved to unit length either by ribozymes formed from both polarities of the viroid genomic RNA or by coopted host RNAses. Many viroid-like small circular RNAs are satellites of plant RNA viruses. Ribozyviruses, represented by human hepatitis delta virus, are larger viroid-like circular RNAs that additionally encode the viral nucleocapsid protein. It has been proposed that viroids are direct descendants of primordial RNA replicons that were present in the hypothetical RNA world. We argue, however, that much later origin of viroids, possibly, from recently discovered mobile genetic elements known as retrozymes, is a far more parsimonious evolutionary scenario. Nevertheless, viroids and viroid-like circular RNAs are minimal replicators that are likely to be close to the theoretical lower limit of replicator size and arguably comprise the paradigm for replicator emergence. Thus, although viroid-like replicators are unlikely to be direct descendants of primordial RNA replicators, the study of the diversity and evolution of these ultimate genetic parasites can yield insights into the earliest stages of the evolution of life.

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Collie, Gavin W., Gary N. Parkinson, Stephen Neidle, Frédéric Rosu, Edwin De Pauw, and Valérie Gabelica. "Electrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures." Journal of the American Chemical Society 132, no. 27 (July 14, 2010): 9328–34. http://dx.doi.org/10.1021/ja100345z.

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Vold, B. S., and C. J. Green. "Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P." Journal of Biological Chemistry 263, no. 28 (October 1988): 14390–96. http://dx.doi.org/10.1016/s0021-9258(18)68232-8.

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Pang, Qishen, Tracy A. Christianson, Winifred Keeble, Tara Koretsky, and Grover C. Bagby. "The Anti-apoptotic Function of Hsp70 in the Interferon-inducible Double-stranded RNA-dependent Protein Kinase-mediated Death Signaling Pathway Requires the Fanconi Anemia Protein, FANCC." Journal of Biological Chemistry 277, no. 51 (October 22, 2002): 49638–43. http://dx.doi.org/10.1074/jbc.m209386200.

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Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents. A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR). Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex. By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR bothin vivoandin vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins. Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR. We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function.

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Tannenbaum, SH, and HR Gralnick. "Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells." Blood 75, no. 11 (June 1, 1990): 2177–84. http://dx.doi.org/10.1182/blood.v75.11.2177.2177.

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Abstract Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

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Tannenbaum, SH, and HR Gralnick. "Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells." Blood 75, no. 11 (June 1, 1990): 2177–84. http://dx.doi.org/10.1182/blood.v75.11.2177.bloodjournal75112177.

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Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

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Woodcock, Clayton B., John R. Horton, Xing Zhang, Robert M. Blumenthal, and Xiaodong Cheng. "Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences." Nucleic Acids Research 48, no. 18 (May 26, 2020): 10034–44. http://dx.doi.org/10.1093/nar/gkaa446.

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Abstract S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle–regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

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Yang, Jin, and Thomas H. Eickbush. "RNA-Induced Changes in the Activity of the Endonuclease Encoded by the R2 Retrotransposable Element." Molecular and Cellular Biology 18, no. 6 (June 1, 1998): 3455–65. http://dx.doi.org/10.1128/mcb.18.6.3455.

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ABSTRACT R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods. The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3′ hydroxyl group generated from this nick to prime reverse transcription of its own RNA. This reaction has been termed target-primed reverse transcription (TPRT). Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA. In this study, more sensitive in vitro assays have enabled further characterization of these reactions. R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths. Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex. While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA. Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl. Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site. The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.

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Pogany, Judit, K. Andrew White, and Peter D. Nagy. "Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication." Journal of Virology 79, no. 8 (April 15, 2005): 4859–69. http://dx.doi.org/10.1128/jvi.79.8.4859-4869.2005.

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ABSTRACT The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C · C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

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Benaud, Christelle, Benoît J. Gentil, Nicole Assard, Magalie Court, Jerome Garin, Christian Delphin, and Jacques Baudier. "AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture." Journal of Cell Biology 164, no. 1 (December 29, 2003): 133–44. http://dx.doi.org/10.1083/jcb.200307098.

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Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca2+-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

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Sundararajan, Anuradha, Bum-Soo Lee, and David J. Garfinkel. "The Rad27 (Fen-1) Nuclease Inhibits Ty1 Mobility in Saccharomyces cerevisiae." Genetics 163, no. 1 (January 1, 2003): 55–67. http://dx.doi.org/10.1093/genetics/163.1.55.

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Abstract Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a posttranslational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.

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Ramezani, Ali. "Assessment of an anti-HIV-1 combination gene therapy strategy using the antisense RNA and multimeric hammerhead ribozymes." Frontiers in Bioscience 11, no. 1 (2006): 2940. http://dx.doi.org/10.2741/2023.

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Yeh, Tien-Shun, and Yan-Hwa Wu Lee. "Assembly of Hepatitis Delta Virus Particles: Package of Multimeric Hepatitis Delta Virus Genomic RNA and Role of Phosphorylation." Virology 249, no. 1 (September 1998): 12–20. http://dx.doi.org/10.1006/viro.1998.9310.

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Kang, Sungmuk, Woori Chae, Sung-Kwan Suh, Woong Jung, Dong-Jo Chang, and Sang Soo Hah. "HER2 RNA Aptamer- and Cell Penetrating Peptide-Mediated Delivery of Multimeric Antisense Strands of siRNAs for Gene Silencing." Bulletin of the Korean Chemical Society 37, no. 9 (August 29, 2016): 1440–44. http://dx.doi.org/10.1002/bkcs.10886.

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Al-Khouri, Anna Maria, and Marvin R. Paule. "A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding." Molecular and Cellular Biology 22, no. 3 (February 1, 2002): 750–61. http://dx.doi.org/10.1128/mcb.22.3.750-761.2002.

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ABSTRACT In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

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Fislová, Tatiana, Benjamin Thomas, Katy M. Graef, and Ervin Fodor. "Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT." Journal of Virology 84, no. 17 (June 23, 2010): 8691–99. http://dx.doi.org/10.1128/jvi.00813-10.

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ABSTRACT The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), α- and β-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

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Yen, Li-Chen, Jia-Teh Liao, Hwei-Jen Lee, Wei-Yuan Chou, Chun-Wei Chen, Yi-Ling Lin, and Ching-Len Liao. "The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion." Journal of Virology 90, no. 3 (November 11, 2015): 1178–89. http://dx.doi.org/10.1128/jvi.02057-15.

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ABSTRACTNS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using atrans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain.IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

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Chen, Hsiao-Wen, Robert N. Rainey, Cynthia E. Balatoni, David W. Dawson, Joshua J. Troke, Sylwia Wasiak, Jason S. Hong, et al. "Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis." Molecular and Cellular Biology 26, no. 22 (September 11, 2006): 8475–87. http://dx.doi.org/10.1128/mcb.01002-06.

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ABSTRACT We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-β-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

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Trikha, Roochi, and David W. Brighty. "Phenotypic analysis of human immunodeficiency virus type 1 Rev trimerization-interface mutants in human cells." Journal of General Virology 86, no. 5 (May 1, 2005): 1509–13. http://dx.doi.org/10.1099/vir.0.80572-0.

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Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable ‘trimerization’ interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.

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Ruedas, Rémi, Soumiya Sankari Muthukumar, Sylvie Kieffer-Jaquinod, François-Xavier Gillet, Daphna Fenel, Grégory Effantin, Thomas Pfannschmidt, Yohann Couté, Robert Blanvillain, and David Cobessi. "Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba." International Journal of Molecular Sciences 23, no. 17 (August 31, 2022): 9922. http://dx.doi.org/10.3390/ijms23179922.

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RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.

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te Velthuis, Aartjan J. W., Sjoerd H. E. van den Worm, and Eric J. Snijder. "The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension." Nucleic Acids Research 40, no. 4 (October 29, 2011): 1737–47. http://dx.doi.org/10.1093/nar/gkr893.

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Lerbs-Mache, S. "The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?" Proceedings of the National Academy of Sciences 90, no. 12 (June 15, 1993): 5509–13. http://dx.doi.org/10.1073/pnas.90.12.5509.

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Journal articles on the topic 'Multimeric RNA'

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