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Judith L Fridovichkeil - One of the best experts on this subject based on the ideXlab platform.
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both kh and non kh domain sequences are required for polyribosome association of scp160p in yeast
Nucleic Acids Research, 2004Co-Authors: Aimin Li, Claudia A Vargas, Melissa A Brykailo, Kimberly K Openo, Anita H. Corbett, Judith L FridovichkeilAbstract:Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound Polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160DN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160DC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studiesfurtherdemonstratethatalthoughtheN-truncated protein remains competent to form RNPs, the Ctruncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome associationasanessentialcomponentofScp160pfunction in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.
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scp160p a multiple kh domain protein is a component of mrnp complexes in yeast
Nucleic Acids Research, 2000Co-Authors: Brian D Lang, Judith L FridovichkeilAbstract:Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with Polyribosomes. Furthermore, we have found that Scp160p is released from Polyribosomes by EDTA in the form of a large complex of ≥1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160-null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.
Alexander S Spirin - One of the best experts on this subject based on the ideXlab platform.
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conformation transitions of eukaryotic Polyribosomes during multi round translation
Nucleic Acids Research, 2015Co-Authors: Zhanna A Afonina, Alexander G Myasnikov, Vladimir A Shirokov, Bruno P Klaholz, Alexander S SpirinAbstract:Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic Polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20–30 translation rounds during 6–8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular Polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear Polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) Polyribosomes and the linear (free-shaped and zigzag-like) Polyribosomes were formed (‘juvenile phase’). The progressive loading of the Polyribosomes with translating ribosomes induced the opening of the circular Polyribosomes and the transformation of a major part of the linear Polyribosomes into the dense 3D helices (‘transitional phase’). After 2 h from the beginning (about 8–10 rounds of translation) this compact form of Polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes (‘steady-state phase’). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical Polyribosomes.
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formation of circular Polyribosomes on eukaryotic mrna without cap structure and poly a tail a cryo electron tomography study
Nucleic Acids Research, 2014Co-Authors: Zhanna A Afonina, Alexander G Myasnikov, Vladimir A Shirokov, Bruno P Klaholz, Alexander S SpirinAbstract:The Polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual Polyribosomes. The achieved level of resolution in the reconstructed Polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating Polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3′-UTR derived from non-coding plasmid sequence, also led to the formation of circular Polyribosomes in similar proportion (40%). Moreover, the Polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5′- and 3′-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular Polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.
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topology of mrna chain in isolated eukaryotic double row Polyribosomes
Biochemistry, 2013Co-Authors: Zh A Afonina, Alexander G Myasnikov, Vladimir A Shirokov, Bruno P Klaholz, Nelli F Khabibullina, Yu A Belorusova, Jeanfrancois Menetret, Victor D Vasiliev, Alexander S SpirinAbstract:In the process of protein synthesis, the translating ribosomes of eukaryotic cells form Polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic Polyribosomes are circles (circular Polyribosomes) and two-stranded formations (so-called double-row Polyribosomes). The “long” (i.e. heavy loaded) Polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular (“col-lapsed rings”), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic Polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in Polyribosomes by marking them with gold nanoparticles (3′-end) and initiating 40S subunits (5′-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in Polyribosomes and mutual orientation of ribosomes in them has shown that the double-row Polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.
Nadal Marc - One of the best experts on this subject based on the ideXlab platform.
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RNA topoisomerase is prevalent in all domains of life and associates with Polyribosomes in animals.
'Oxford University Press (OUP)', 2016Co-Authors: Ahmad Muzammil, Xu Yutong, Lee, Seung Kyu, Martindale, Jennifer L, Shen Weiping, Li Wen, Zou Sige, Ciaramella Maria, Debat Hélène, Nadal MarcAbstract:International audienceDNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with Polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of Polyribosomes in animals
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RNA topoisomerase is prevalent in all domains of life and associates with Polyribosomes in animals
NUCLEIC ACIDS RESEARCH, 2016Co-Authors: Ahmad Muzammil, Xu Yutong, Lee, Seung Kyu, Martindale, Jennifer L, Shen Weiping, Li Wen, Zou Sige, Ciaramella Maria, Debat Hélène, Nadal MarcAbstract:DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl- terminal domain (CTD),which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3 beta differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3 beta proteins from several animals associate with Polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3 beta-polyribosome association requires TDRD3, which directly interacts with Top3 beta and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of Polyribosomes in animals.Intramural Research Program of the National Institute on Aging [Z01 AG000657-08]; National Cancer Institute [Z01 BC006161]; National Institutes of Health; National Basic Research Program of China [2013CB911002]; National Natural Science Foundation of China [31271435]; National Institute on Aging, NIH, Intramural fundingSCI(E)PubMedARTICLExudongyi@pku.edu.cn; wangw@grc.nia.nih.gov136335-63494
Zhanna A Afonina - One of the best experts on this subject based on the ideXlab platform.
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conformation transitions of eukaryotic Polyribosomes during multi round translation
Nucleic Acids Research, 2015Co-Authors: Zhanna A Afonina, Alexander G Myasnikov, Vladimir A Shirokov, Bruno P Klaholz, Alexander S SpirinAbstract:Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic Polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20–30 translation rounds during 6–8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular Polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear Polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) Polyribosomes and the linear (free-shaped and zigzag-like) Polyribosomes were formed (‘juvenile phase’). The progressive loading of the Polyribosomes with translating ribosomes induced the opening of the circular Polyribosomes and the transformation of a major part of the linear Polyribosomes into the dense 3D helices (‘transitional phase’). After 2 h from the beginning (about 8–10 rounds of translation) this compact form of Polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes (‘steady-state phase’). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical Polyribosomes.
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formation of circular Polyribosomes on eukaryotic mrna without cap structure and poly a tail a cryo electron tomography study
Nucleic Acids Research, 2014Co-Authors: Zhanna A Afonina, Alexander G Myasnikov, Vladimir A Shirokov, Bruno P Klaholz, Alexander S SpirinAbstract:The Polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual Polyribosomes. The achieved level of resolution in the reconstructed Polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating Polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3′-UTR derived from non-coding plasmid sequence, also led to the formation of circular Polyribosomes in similar proportion (40%). Moreover, the Polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5′- and 3′-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular Polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.
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during multi-round translation
2014Co-Authors: Zhanna A Afonina, Vladimir A Shirokov, Bruno P Klaholz, Er G. Myasnikov, Er S. SpirinAbstract:Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyri-bosomes formed in a long-term cell-free translation system were analyzed over all the active system life-time (20–30 translation rounds during 6–8 h in wheat germ extract at 25◦C). Three distinct types of the conformations were observed: (i) circular polyribo-somes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear Polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) Polyribosomes and the linear (free-shaped and zigzag-like) Polyribosomes were formed (‘juvenile phase’). The progressive load-ing of the Polyribosomes with translating ribosomes induced the opening of the circular Polyribosomes and the transformation of a major part of the lin-ear Polyribosomes into the dense 3D helices (‘transi-tional phase’). After 2 h from the beginning (about 8–10 rounds of translation) this compact form of Polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes (‘steady-state phase’). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical Polyribosomes
C.r Benech - One of the best experts on this subject based on the ideXlab platform.
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Ribosomes and Polyribosomes are present in the squid giant axon: an immunocytochemical study.
Neuroscience, 1999Co-Authors: J.r Sotelo, Alejandra Kun, Juan Claudio Benech, A Giuditta, J Morillas, C.r BenechAbstract:Ribosomes and Polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and Polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and Polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.