Journal articles on the topic 'Ribonucleoprotein particles RNPs'
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1
Peek, R., G. J. Pruijn, A. J. van der Kemp, and W. J. van Venrooij. "Subcellular distribution of Ro ribonucleoprotein complexes and their constituents." Journal of Cell Science 106, no. 3 (November 1, 1993): 929–35. http://dx.doi.org/10.1242/jcs.106.3.929.
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Ro ribonucleoprotein particles (Ro RNPs) are complexes of several proteins with a small RNA polymerase III-transcribed Ro RNA. Despite their relative abundance and evolutionary conservation no function has as yet been ascribed to these complexes. Also their subcellular distribution is still largely unknown as immunofluorescence studies concerning their localization have produced conflicting data. We have used cell enucleation to fractionate cells into cytoplasmic and nuclear fractions. Analysis of these fractions revealed an exclusively cytoplasmic localization for the Ro RNPs. The majority of the Ro RNAs are shown to be stably associated with all three known Ro RNP proteins. Although no Ro RNAs could be detected in the nuclear fraction, the Ro RNP-specific proteins were abundantly present. These nuclear non-Ro RNA-associated proteins are shown to be capable of binding Ro RNAs.
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Liang, Bo, and Hong Li. "Structures of ribonucleoprotein particle modification enzymes." Quarterly Reviews of Biophysics 44, no. 1 (November 26, 2010): 95–122. http://dx.doi.org/10.1017/s0033583510000235.
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AbstractSmall nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo.
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Simons, F. H., G. J. Pruijn, and W. J. van Venrooij. "Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes." Journal of Cell Biology 125, no. 5 (June 1, 1994): 981–88. http://dx.doi.org/10.1083/jcb.125.5.981.
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Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.
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Peek, R., G. J. Pruijn, and W. J. van Venrooij. "Epitope specificity determines the ability of anti-Ro52 autoantibodies to precipitate Ro ribonucleoprotein particles." Journal of Immunology 153, no. 9 (November 1, 1994): 4321–29. http://dx.doi.org/10.4049/jimmunol.153.9.4321.
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Abstract Ro ribonucleoprotein particles (Ro RNPs) are evolutionarily conserved cytoplasmic complexes of unknown function. They are composed of several proteins and a small, RNA polymerase III-transcribed Ro or Y RNA. Abs directed against the protein moiety of Ro RNPs are often found in sera of patients suffering from certain autoimmune disorders. The association of one of the Ro proteins, a protein of 52 kDa (Ro52), with Ro RNPs is still questionable. In this study, we have used anti-Ro52 Abs isolated from autoimmune sera to locate the antigenic determinants of Ro52 and to analyze the correlation between regions of Ro52 recognized by these Abs and their ability to immunoprecipitate Ro RNPs. The results indicate that the autoimmune response against Ro52 is heterogeneous and that the exclusive recognition of certain epitopes does not result in immunoprecipitation of Ro RNPs.
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Hall, Kathleen B. "RNA and Proteins: Mutual Respect." F1000Research 6 (March 27, 2017): 345. http://dx.doi.org/10.12688/f1000research.10572.1.
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Proteins and RNA are often found in ribonucleoprotein particles (RNPs), where they function in cellular processes to synthesize proteins (the ribosome), chemically modify RNAs (small nucleolar RNPs), splice pre-mRNAs (the spliceosome), and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli). Each RNA–protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.
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Bachmann, M., W. J. Mayet, H. C. Schröder, K. Pfeifer, K. H. Meyer zum Büschenfelde, and W. E. G. Müller. "Identification of the Ro and La antigens in the endoribonuclease VII–ribonucleoprotein complex." Biochemical Journal 243, no. 1 (April 1, 1987): 189–94. http://dx.doi.org/10.1042/bj2430189.
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45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.
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Wurtz-T, E. Kiseleva, G. Nacheva, A. Alzhanova-Ericcson, A. Rosén, and B. Daneholt. "Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles." Molecular and Cellular Biology 16, no. 4 (April 1996): 1425–35. http://dx.doi.org/10.1128/mcb.16.4.1425.
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Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.
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Zillmann, M., M. L. Zapp, and S. M. Berget. "Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles." Molecular and Cellular Biology 8, no. 2 (February 1988): 814–21. http://dx.doi.org/10.1128/mcb.8.2.814-821.1988.
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Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.
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Zillmann, M., M. L. Zapp, and S. M. Berget. "Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles." Molecular and Cellular Biology 8, no. 2 (February 1988): 814–21. http://dx.doi.org/10.1128/mcb.8.2.814.
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Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.
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Kedersha, Nancy L., and Leonard H. Rome. "Immunolocalization of vault particles in cultured cells." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 458–59. http://dx.doi.org/10.1017/s0424820100122691.
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We first reported on the existence of vault ribonucleoprotein particles in 1986, and since this study we have demonstrated that these unusual RNPs are ubiquitously expressed and highly conserved among diverse eukaryotes. These particles are quite large (65 x 35 nm) and distinctly regular in shape and dimensions. The polypeptide composition of vaults is also similar between species, dominated by a∼100 Kd protein which makes up >70% of the particles mass. The RNA component of vaults, which has been sequenced and characterized from both rat and bullfrog, does not appear to serve a structural role, and due to the strong conservation of its secondary structure could serve a functional role.
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Liu, Chengyu, and L. Dennis Smith. "In vivo storage of XR family interspersed RNA in Xenopus oocytes." Zygote 3, no. 1 (February 1995): 37–44. http://dx.doi.org/10.1017/s0967199400002367.
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SummaryInterspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNa (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. Im small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentaion constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentaion constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis.
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Patton, J. R. "Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro." Molecular and Cellular Biology 11, no. 12 (December 1991): 5998–6006. http://dx.doi.org/10.1128/mcb.11.12.5998-6006.1991.
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The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.
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Patton, J. R. "Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro." Molecular and Cellular Biology 11, no. 12 (December 1991): 5998–6006. http://dx.doi.org/10.1128/mcb.11.12.5998.
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The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.
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Kuhn-Holsken, E., O. Dybkov, B. Sander, R. Luhrmann, and H. Urlaub. "Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry." Nucleic Acids Research 35, no. 15 (July 11, 2007): e95-e95. http://dx.doi.org/10.1093/nar/gkm540.
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García-Rodríguez, Fernando M., José L. Neira, Marco Marcia, María D. Molina-Sánchez, and Nicolás Toro. "A group II intron-encoded protein interacts with the cellular replicative machinery through the β-sliding clamp." Nucleic Acids Research 47, no. 14 (May 25, 2019): 7605–17. http://dx.doi.org/10.1093/nar/gkz468.
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Abstract Group II introns are self-splicing mobile genetic retroelements. The spliced intron RNA and the intron-encoded protein (IEP) form ribonucleoprotein particles (RNPs) that recognize and invade specific DNA target sites. The IEP is a reverse transcriptase/maturase that may bear a C-terminal endonuclease domain enabling the RNP to cleave the target DNA strand to prime reverse transcription. However, some mobile introns, such as RmInt1, lack the En domain but nevertheless retrohome efficiently to transient single-stranded DNA target sites at a DNA replication fork. Their mobility is associated with host DNA replication, and they use the nascent lagging strand as a primer for reverse transcription. We searched for proteins that interact with RmInt1 RNPs and direct these RNPs to the DNA replication fork. Co-immunoprecipitation assays suggested that DnaN (the β-sliding clamp), a component of DNA polymerase III, interacts with the protein component of the RmInt1 RNP. Pulldown assays, far-western blots and biolayer interferometry supported this interaction. Peptide binding assays also identified a putative DnaN-interacting motif in the RmInt1 IEP structurally conserved in group II intron IEPs. Our results suggest that intron RNP interacts with the β-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks.
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Lyu, Pin, Parisa Javidi-Parsijani, Anthony Atala, and Baisong Lu. "Delivering Cas9/sgRNA ribonucleoprotein (RNP) by lentiviral capsid-based bionanoparticles for efficient ‘hit-and-run’ genome editing." Nucleic Acids Research 47, no. 17 (July 12, 2019): e99-e99. http://dx.doi.org/10.1093/nar/gkz605.
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AbstractTransient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.
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Patel, Snehal Bhikhu, Natalya Novikova, and Michel Bellini. "Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts." Journal of Cell Biology 178, no. 6 (September 10, 2007): 937–49. http://dx.doi.org/10.1083/jcb.200706134.
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In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.
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Petri, Sebastian, Matthias Grimmler, Sabine Over, Utz Fischer, and Oliver J. Gruss. "Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2Cγ governs Cajal body localization and stability of the SMN complex." Journal of Cell Biology 179, no. 3 (November 5, 2007): 451–65. http://dx.doi.org/10.1083/jcb.200704163.
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The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cγ interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex.
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Tseng, Chi-Kang. "Study of Telomere and Telomerase Dynamics." Impact 2021, no. 8 (October 28, 2021): 22–24. http://dx.doi.org/10.21820/23987073.2021.8.22.
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Telomerases play a key role in maintaining the necessary level of chromosomal degradation in all cells and so they are an important target for in depth molecular, genetic, and in vivo investigations. Assistant Professor Chi-Kang Tseng, Graduate Institute of Microbiology, National Taiwan University College of Medicine (NTUCM), Taiwan, specialises in the molecular biology of ribonucleoprotein particles (RNPs), primarily telomerases and spliceosomes and collaborates with clinicians and experts in related fields to ensure his work is as relevant as possible and can potentially be directly translated into a clinical setting. A key focus for Tseng is on the precise molecular biology behind the function and dynamics of RNPs. He and his team conduct in vivo experimentation in cell cultures of induced pluripotent stem cells, and also CRISPR-based gene editing techniques. In addition, the researchers have developed a novel technique for the in vitro analysis of telomerase biogenesis. Tseng and the team are also exploring the action and dynamics of the spliceosome. The researchers' work has direct relevance to four key diseases that are associated with telomerases and/or the spliceosome: Myelodysplastic syndromes; Dyskeratosis Congenita; Parkinson's disease; and cancer.
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Li, Hai-Ou, Ya-Feng Zhu, Makoto Asakawa, Hidekazu Kuma, Takahiro Hirata, Yasuji Ueda, Yun-Sik Lee, et al. "A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression." Journal of Virology 74, no. 14 (July 15, 2000): 6564–69. http://dx.doi.org/10.1128/jvi.74.14.6564-6569.2000.
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ABSTRACT We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of theParamyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.
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Severt, W. L., T. U. Biber, X. Wu, N. B. Hecht, R. J. DeLorenzo, and E. R. Jakoi. "The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites." Journal of Cell Science 112, no. 21 (November 1, 1999): 3691–702. http://dx.doi.org/10.1242/jcs.112.21.3691.
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Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.
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Mische, Sarah, Mingang Li, Madeline Serr, and Thomas S. Hays. "Direct Observation of Regulated Ribonucleoprotein Transport Across the Nurse Cell/Oocyte Boundary." Molecular Biology of the Cell 18, no. 6 (June 2007): 2254–63. http://dx.doi.org/10.1091/mbc.e06-10-0959.
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In Drosophila, the asymmetric localization of specific mRNAs to discrete regions within the developing oocyte determines the embryonic axes. The microtubule motors dynein and kinesin are required for the proper localization of the determinant ribonucleoprotein (RNP) complexes, but the mechanisms that account for RNP transport to and within the oocyte are not well understood. In this work, we focus on the transport of RNA complexes containing bicoid (bcd), an anterior determinant. We show in live egg chambers that, within the nurse cell compartment, dynein actively transports green fluorescent protein-tagged Exuperantia, a cofactor required for bcd RNP localization. Surprisingly, the loss of kinesin I activity elevates RNP motility in nurse cells, whereas disruption of dynein activity inhibits RNP transport. Once RNPs are transferred through the ring canal to the oocyte, they no longer display rapid, linear movements, but they are distributed by cytoplasmic streaming and gradually disassemble. By contrast, bcd mRNA injected into oocytes assembles de novo into RNP particles that exhibit rapid, dynein-dependent transport. We speculate that after delivery to the oocyte, RNP complexes may disassemble and be remodeled with appropriate accessory factors to ensure proper localization.
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Liu, Teresa, and Zhiping Ye. "Introduction of a Temperature-Sensitive Phenotype into Influenza A/WSN/33 Virus by Altering the Basic Amino Acid Domain of Influenza Virus Matrix Protein." Journal of Virology 78, no. 18 (September 15, 2004): 9585–91. http://dx.doi.org/10.1128/jvi.78.18.9585-9591.2004.
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ABSTRACT Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.
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Yang, Pok Kwan, Coralie Hoareau, Carine Froment, Bernard Monsarrat, Yves Henry, and Guillaume Chanfreau. "Cotranscriptional Recruitment of the Pseudouridylsynthetase Cbf5p and of the RNA Binding Protein Naf1p during H/ACA snoRNP Assembly." Molecular and Cellular Biology 25, no. 8 (April 15, 2005): 3295–304. http://dx.doi.org/10.1128/mcb.25.8.3295-3304.2005.
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ABSTRACT H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) are essential for the maturation and pseudouridylation of the precursor of rRNAs and other stable RNAs. Although the RNA and protein components of these RNPs have been identified, the mechanisms by which they are assembled in vivo are poorly understood. Here we show that the RNA binding protein Naf1p, which is required for H/ACA snoRNPs stability, associates with RNA polymerase II-associated proteins Spt16p, Tfg1p, and Sub1p and with H/ACA snoRNP proteins. Chromatin immunoprecipitation experiments show that Naf1p and the pseudouridylsynthetase Cbf5p cross-link specifically with the chromatin of H/ACA small nucleolar RNA (snoRNA) genes. Naf1p and Cbf5p cross-link predominantly with the 3′ end of these genes, in a pattern similar to that observed for transcription elongation factor Spt16p. Cross-linking of Naf1p to H/ACA snoRNA genes requires active transcription and intact H/ACA snoRNA sequences but does not require the RNA polymerase II CTD kinase Ctk1p. These results suggest that Naf1p and Cbf5p are recruited in a cotranscriptional manner during H/ACA snoRNP assembly, possibly by binding to the nascent H/ACA snoRNA transcript during elongation or termination of transcription of H/ACA snoRNA genes.
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Finke, Stefan, Krzysztof Brzózka, and Karl-Klaus Conzelmann. "Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles." Journal of Virology 78, no. 22 (November 15, 2004): 12333–43. http://dx.doi.org/10.1128/jvi.78.22.12333-12343.2004.
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ABSTRACT Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ΔP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.
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Dez, Christophe, Jacqueline Noaillac-Depeyre, Michèle Caizergues-Ferrer, and Yves Henry. "Naf1p, an Essential Nucleoplasmic Factor Specifically Required for Accumulation of Box H/ACA Small Nucleolar RNPs." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 7053–65. http://dx.doi.org/10.1128/mcb.22.20.7053-7065.2002.
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ABSTRACT Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. The ways in which these particles are assembled and correctly localized in the dense fibrillar component of the nucleolus remain largely unknown. Recently, the essential Saccharomyces cerevisiae Naf1p protein (encoded by the YNL124W open reading frame) was found to interact in a two-hybrid assay with two core protein components of mature H/ACA snoRNPs, Cbf5p and Nhp2p (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2001). Here we show that several H/ACA snoRNP components are weakly but specifically immunoprecipitated with epitope-tagged Naf1p, suggesting that the latter protein is involved in H/ACA snoRNP biogenesis, trafficking, and/or function. Consistent with this, we find that depletion of Naf1p leads to a defect in 18S rRNA accumulation. Naf1p is unlikely to directly assist H/ACA snoRNPs during pre-rRNA processing in the dense fibrillar component of the nucleolus for two reasons. Firstly, Naf1p accumulates predominantly in the nucleoplasm. Secondly, Naf1p sediments in a sucrose gradient chiefly as a free protein or associated in a complex of the size of free snoRNPs, whereas extremely little Naf1p is found in fractions containing preribosomes. These results are more consistent with a role for Naf1p in H/ACA snoRNP biogenesis and/or intranuclear trafficking. Indeed, depletion of Naf1p leads to a specific and dramatic decrease in the steady-state accumulation of all box H/ACA snoRNAs tested and of Cbf5p, Gar1p, and Nop10p. Naf1p is unlikely to be directly required for the synthesis of H/ACA snoRNP components. Naf1p could participate in H/ACA snoRNP assembly and/or transport.
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Glätzer, K. H., and P. M. Kloetzel. "Differential chromosomal distribution of ribonucleoprotein antigens in nuclei of Drosophila spermatocytes." Journal of Cell Biology 103, no. 6 (December 1, 1986): 2113–19. http://dx.doi.org/10.1083/jcb.103.6.2113.
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The ribonucleoprotein (RNP) composition of the active Y chromosomal structures in spermatocyte nuclei of Drosophila hydei has been investigated using the anti-RNP antibodies Dm 28K2 and pp60 as a probe. Antibody Dm 28K2 was raised against an RNP protein of cytoplasmic RNP particles in D. melanogaster cells, while antibody pp60 was raised against a pre-messenger RNP fraction from oocytes of Xenopus laevis. Both antibodies detect nuclear RNP (nRNP) antigens of D. hydei. This is shown by CsCl density centrifugation of nRNP from D. hydei cells and immunoblotting across the density gradient. Dm 28K2 and pp60 recognize antigens of nRNP complexes which band at a characteristic buoyant density of approximately 1.4 g/cm3 in CsCl. By indirect immunofluorescence we observe that the nRNP complexes identified by Dm 28K2 are localized at only two of the five Y chromosomal loop structures which are named according to their distinct morphology. Dm 28K2 decorates RNPs within the "clubs," within the cones, and within the matrix of the "pseudonucleolus." Ultrastructural bodies that are candidates for this immunoreaction are RNP granules that resemble the so-called perichromatin granules. Antibody pp60 recognizes RNP complexes close to the axes of the active Y chromatin. In the "pseudonucleolus" it can be shown that the structures recognized by pp60 are quite distinct from those detected by Dm 28K2. Thus, the "pseudonucleolus" is a striking example for the presence of different RNP populations within a same defined nuclear compartment. Together with previous results (Glätzer, K. H., 1984, Mol. Gen. Genet., 196:236-243), our data represent evidence that the morphological and apparently functional differences between the active Y chromosomal loops, which are involved in male fertility, are caused by the presence of qualitatively and possibly also functionally different RNP populations within these nuclear compartments. Because both RNP antigens are discussed in the literature in connection with repressed mRNP the observed cross-reaction of the respective antibodies in D. hydei suggests a more general and important function of these proteins in the RNA metabolism of eukaryotic cells.
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Giorgini, Flaviano, Holly G. Davies, and Robert E. Braun. "MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo." Molecular and Cellular Biology 21, no. 20 (October 15, 2001): 7010–19. http://dx.doi.org/10.1128/mcb.21.20.7010-7019.2001.
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ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.
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Burleigh, Laura M., Lesley J. Calder, John J. Skehel, and David A. Steinhauer. "Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes." Journal of Virology 79, no. 2 (January 15, 2005): 1262–70. http://dx.doi.org/10.1128/jvi.79.2.1262-1270.2005.
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ABSTRACT Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated “helix six.” This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.
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Schmitt, Phuong Tieu, Greeshma Ray, and Anthony P. Schmitt. "The C-Terminal End of Parainfluenza Virus 5 NP Protein Is Important for Virus-Like Particle Production and M-NP Protein Interaction." Journal of Virology 84, no. 24 (October 13, 2010): 12810–23. http://dx.doi.org/10.1128/jvi.01885-10.
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ABSTRACT Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI−. Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.
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Brown, Gaie, James Aitken, Helen W. McL Rixon, and Richard J. Sugrue. "Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells." Journal of General Virology 83, no. 3 (March 1, 2002): 611–21. http://dx.doi.org/10.1099/0022-1317-83-3-611.
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We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.
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Chan, Wai-Hon, Andy Ka-Leung Ng, Nicole C. Robb, Mandy Ka-Han Lam, Paul Kay-Sheung Chan, Shannon Wing-Ngor Au, Jia-Huai Wang, Ervin Fodor, and Pang-Chui Shaw. "Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities." Journal of Virology 84, no. 14 (May 12, 2010): 7337–45. http://dx.doi.org/10.1128/jvi.02474-09.
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ABSTRACT Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.
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Agredano-Moreno, Lourdes-Teresa, María De Lourdes Segura-Valdez, Jaime Jiménez-Ramírez, and Luis-Felipe Jiménez-García. "Lacandonia granules are present in the cell nucleus of Welwitschia mirabilis." Botanical Sciences 96, no. 4 (November 29, 2018): 678. http://dx.doi.org/10.17129/botsci.1924.
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<p><strong>Background:</strong> <em>Lacandonia</em> granules are extranucleolar ribonucleoprotein (RNPs) particles, 32 nanometers in diameter that were first described in the nucleus of <em>Lacandonia schismatica</em>. Cytochemical and immunocytochemical studies suggest that these particles are equivalent to perichromatin and Balbiani ring granules described in mammals and salivary glands cells of the insect <em>Chironomus tentans, </em>respectively. <em>Lacandonia</em> granules are also present in the related <em>Triuris</em> <em>brevystilis</em>,<em> </em>and they were later described in the gymnosperm <em>Ginkgo biloba</em>. These findings suggest that <em>Lacandonia </em>granules have a wider distribution in the plant kingdom. </p><p><strong>Species study:</strong> The plant <em>Welwitschia mirabilis, </em>a gymnosperm of the order Gnetales.</p><p><strong>Hyphotesis: </strong><em>Lacandonia</em> granules are present in the cell nucleus of <em>W. mirabilis</em>.<strong> </strong></p><p><strong>Methods:</strong> Plants were cultivated<strong> </strong>in a germination chamber and samples of leaves were processed for transmission electron microscopy. Thin sections were stained with the EDTA technique preferential for ribonucleoproteins and osmium amine specific for DNA and observed with a microscopy.</p><p><strong>Results:</strong> Light, electronic and atomic force microscopy revealed that cell nuclei of <em>W. mirabilis</em> display a reticulated arrangement of chromatin. Moreover, granules of 32.17 ± 1.7 nm in diameter were observed among strands of reticulated chromatin.</p><p><strong>Conclusions:</strong> Our results indicate that <em>Lacandonia</em> granules are present in the nuclei of the gnetal <em>W. </em> <em>mirabilis.</em></p>
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Carotenuto, Pietro, Annalisa Pecoraro, Gaetano Palma, Giulia Russo, and Annapina Russo. "Therapeutic Approaches Targeting Nucleolus in Cancer." Cells 8, no. 9 (September 16, 2019): 1090. http://dx.doi.org/10.3390/cells8091090.
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The nucleolus is a distinct sub-cellular compartment structure in the nucleus. First observed more than 200 years ago, the nucleolus is detectable by microscopy in eukaryotic cells and visible during the interphase as a sub-nuclear structure immersed in the nucleoplasm, from which it is not separated from any membrane. A huge number of studies, spanning over a century, have identified ribosome biogenesis as the main function of the nucleolus. Recently, novel functions, independent from ribosome biogenesis, have been proposed by several proteomic, genomic, and functional studies. Several works have confirmed the non-canonical role for nucleoli in regulating important cellular processes including genome stability, cell-cycle control, the cellular senescence, stress responses, and biogenesis of ribonucleoprotein particles (RNPs). Many authors have shown that both canonical and non-canonical functions of the nucleolus are associated with several cancer-related processes. The association between the nucleolus and cancer, first proposed by cytological and histopathological studies showing that the number and shape of nucleoli are commonly altered in almost any type of cancer, has been confirmed at the molecular level by several authors who demonstrated that numerous mechanisms occurring in the nucleolus are altered in tumors. Recently, therapeutic approaches targeting the nucleolus in cancer have started to be considered as an emerging “hallmark” of cancer and several therapeutic interventions have been developed. This review proposes an up-to-date overview of available strategies targeting the nucleolus, focusing on novel targeted therapeutic approaches. Finally, a target-based classification of currently available treatment will be proposed.
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Karpova, O. V., O. V. Zayakina, M. V. Arkhipenko, E. V. Sheval, O. I. Kiselyova, V. Yu Poljakov, I. V. Yaminsky, N. P. Rodionova, and J. G. Atabekov. "Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’ complexes." Journal of General Virology 87, no. 9 (September 1, 2006): 2731–40. http://dx.doi.org/10.1099/vir.0.81993-0.
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Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5′-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP–RNA interactions resulting in head formation prevailed over TGBp1–RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5′ end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP–RNA STPs are produced, due to encapsidation of the 5′-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.
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Surtees, Rebecca, Stuart D. Dowall, Amelia Shaw, Stuart Armstrong, Roger Hewson, Miles W. Carroll, Jamel Mankouri, Thomas A. Edwards, Julian A. Hiscox, and John N. Barr. "Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle." Journal of Virology 90, no. 20 (August 10, 2016): 9305–16. http://dx.doi.org/10.1128/jvi.00661-16.
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ABSTRACTTheNairovirusgenus of theBunyaviridaefamily contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease.IMPORTANCENairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance.
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Boulon, Séverine, Nathalie Marmier-Gourrier, Bérengère Pradet-Balade, Laurence Wurth, Céline Verheggen, Beáta E. Jády, Benjamin Rothé, et al. "The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery." Journal of Cell Biology 180, no. 3 (February 11, 2008): 579–95. http://dx.doi.org/10.1083/jcb.200708110.
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RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.
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Yean, S. L., and R. J. Lin. "U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction." Molecular and Cellular Biology 11, no. 11 (November 1991): 5571–77. http://dx.doi.org/10.1128/mcb.11.11.5571-5577.1991.
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U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.
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Yean, S. L., and R. J. Lin. "U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction." Molecular and Cellular Biology 11, no. 11 (November 1991): 5571–77. http://dx.doi.org/10.1128/mcb.11.11.5571.
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U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.
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Malhotra, A., R. K. Tan, and S. C. Harvey. "Modeling large RNAs and ribonucleoprotein particles using molecular mechanics techniques." Biophysical Journal 66, no. 6 (June 1994): 1777–95. http://dx.doi.org/10.1016/s0006-3495(94)80972-5.
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Överby, Anna K., Vsevolod Popov, Etienne P. A. Neve, and Ralf F. Pettersson. "Generation and Analysis of Infectious Virus-Like Particles of Uukuniemi Virus (Bunyaviridae): a Useful System for Studying Bunyaviral Packaging and Budding." Journal of Virology 80, no. 21 (August 23, 2006): 10428–35. http://dx.doi.org/10.1128/jvi.01362-06.
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ABSTRACT In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.
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Galardi, Silvia, Alessandro Fatica, Angela Bachi, Andrea Scaloni, Carlo Presutti, and Irene Bozzoni. "Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro." Molecular and Cellular Biology 22, no. 19 (October 1, 2002): 6663–68. http://dx.doi.org/10.1128/mcb.22.19.6663-6668.2002.
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ABSTRACT Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity.
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de Lucas, Susana, Joan Peredo, Rosa María Marión, Carmen Sánchez, and Juan Ortín. "Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication." Journal of Virology 84, no. 15 (May 26, 2010): 7603–12. http://dx.doi.org/10.1128/jvi.00504-10.
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ABSTRACT The influenza A virus genome consists of 8 negative-stranded RNA segments. NS1 is a nonstructural protein that participates in different steps of the virus infectious cycle, including transcription, replication, and morphogenesis, and acts as a virulence factor. Human Staufen1 (hStau1), a protein involved in the transport and regulated translation of cellular mRNAs, was previously identified as a NS1-interacting factor. To investigate the possible role of hStau1 in the influenza virus infection, we characterized the composition of hStau1-containing granules isolated from virus-infected cells. Viral NS1 protein and ribonucleoproteins (RNPs) were identified in these complexes by Western blotting, and viral mRNAs and viral RNAs (vRNAs) were detected by reverse transcription (RT)-PCR. Also, colocalization of hStau1 with NS1, nucleoprotein (NP), and PA in the cytosol of virus-infected cells was shown by immunofluorescence. To analyze the role of hStau1 in the infection, we downregulated its expression by gene silencing. Human HEK293T cells or A549 cells were silenced using either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting four independent sites in the hStau1 mRNA. The yield of influenza virus was reduced 5 to 10 times in the various hStau1-silenced cells compared to that in control silenced cells. The expression levels of viral proteins and their nucleocytoplasmic localization were not affected upon hStau1 silencing, but virus particle production, as determined by purification of virions from supernatants, was reduced. These results indicate a role for hStau1 in late events of the influenza virus infection, possibly during virus morphogenesis.
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Martinez-Salas, Encarnacion, Azman Embarc-Buh, and Rosario Francisco-Velilla. "Emerging Roles of Gemin5: From snRNPs Assembly to Translation Control." International Journal of Molecular Sciences 21, no. 11 (May 29, 2020): 3868. http://dx.doi.org/10.3390/ijms21113868.
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RNA-binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The disfunction of RBPs is frequently the cause of cell disorders which are incompatible with life. Furthermore, the ordered assembly of RBPs and RNAs in ribonucleoprotein (RNP) particles determines the function of biological complexes, as illustrated by the survival of the motor neuron (SMN) complex. Defects in the SMN complex assembly causes spinal muscular atrophy (SMA), an infant invalidating disease. This multi-subunit chaperone controls the assembly of small nuclear ribonucleoproteins (snRNPs), which are the critical components of the splicing machinery. However, the functional and structural characterization of individual members of the SMN complex, such as SMN, Gemin3, and Gemin5, have accumulated evidence for the additional roles of these proteins, unveiling their participation in other RNA-mediated events. In particular, Gemin5 is a multidomain protein that comprises tryptophan-aspartic acid (WD) repeat motifs at the N-terminal region, a dimerization domain at the middle region, and a non-canonical RNA-binding domain at the C-terminal end of the protein. Beyond small nuclear RNA (snRNA) recognition, Gemin5 interacts with a selective group of mRNA targets in the cell environment and plays a key role in reprogramming translation depending on the RNA partner and the cellular conditions. Here, we review recent studies on the SMN complex, with emphasis on the individual components regarding their involvement in cellular processes critical for cell survival.
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Spann, P., M. Feinerman, J. Sperling, and R. Sperling. "Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs." Proceedings of the National Academy of Sciences 86, no. 2 (January 1, 1989): 466–70. http://dx.doi.org/10.1073/pnas.86.2.466.
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McNally, K. P., and N. Agabian. "Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo." Molecular and Cellular Biology 12, no. 11 (November 1992): 4844–51. http://dx.doi.org/10.1128/mcb.12.11.4844-4851.1992.
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The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.
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McNally, K. P., and N. Agabian. "Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo." Molecular and Cellular Biology 12, no. 11 (November 1992): 4844–51. http://dx.doi.org/10.1128/mcb.12.11.4844.
Full textAbstract:
The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.
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Leung, Eileen, and Jeremy D. Brown. "Biogenesis of the signal recognition particle." Biochemical Society Transactions 38, no. 4 (July 26, 2010): 1093–98. http://dx.doi.org/10.1042/bst0381093.
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Assembly of ribonucleoprotein complexes is a facilitated quality-controlled process that typically includes modification to the RNA component from precursor to mature form. The SRP (signal recognition particle) is a cytosolic ribonucleoprotein that catalyses protein targeting to the endoplasmic reticulum. Assembly of SRP is largely nucleolar, and most of its protein components are required to generate a stable complex. A pre-SRP is exported from the nucleus to the cytoplasm where the final protein, Srp54p, is incorporated. Although this outline of the SRP assembly pathway has been determined, factors that facilitate this and/or function in quality control of the RNA are poorly understood. In the present paper, the SRP assembly pathway is summarized, and evidence for the involvement of both the Rex1p and nuclear exosome nucleases and the TRAMP (Trf4–Air2–Mtr4p polyadenylation) adenylase in quality control of SRP RNA is discussed. The RNA component of SRP is transcribed by RNA polymerase III, and both La, which binds all newly transcribed RNAs generated by this enzyme, and the nuclear Lsm complex are implicated in SRP RNA metabolism.
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Wang, H., J. C. Kennell, M. T. Kuiper, J. R. Sabourin, R. Saldanha, and A. M. Lambowitz. "The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA." Molecular and Cellular Biology 12, no. 11 (November 1992): 5131–44. http://dx.doi.org/10.1128/mcb.12.11.5131-5144.1992.
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The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.
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Wang, H., J. C. Kennell, M. T. Kuiper, J. R. Sabourin, R. Saldanha, and A. M. Lambowitz. "The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA." Molecular and Cellular Biology 12, no. 11 (November 1992): 5131–44. http://dx.doi.org/10.1128/mcb.12.11.5131.
Full textAbstract:
The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.