scholarly journals Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies

1992 ◽  
Vol 117 (1) ◽  
pp. 1-14 ◽  
Author(s):  
M Carmo-Fonseca ◽  
R Pepperkok ◽  
MT Carvalho ◽  
AI Lamond
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Splicing Factor ◽  
Transcription Inhibitors ◽  
Coiled Bodies ◽  
Protein Components ◽  
Nascent Transcripts ◽  
Labeling Method ◽  
Alpha Amanitin

We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the U1, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A. Merdes, C. Brunner, P. D. Zamore, M. R. Green, E. Hurt, and A. I. Lamond. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:195-206; Carmo-Fonseca, M., R. Pepperkok, B. S. Sproat, W. Ansorge, M. S. Swanson, and A. I. Lamond. 1991 EMBO (Eur. Mol. Biol. Organ.) J. 10:1863-1873). Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors alpha-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock. This indicates that the association of snRNPs with coiled bodies may be connected with the metabolism of nascent transcripts. A novel labeling method is described which shows both the RNA and protein components of individual snRNPs colocalizing in situ. Using this procedure all spliceosomal snRNPs are seen distributed in a nonhomogeneous pattern throughout the nucleoplasm, excluding nucleoli. They are most concentrated in coiled bodies, but in addition are present in "speckled" structures which are distinct from coiled bodies and which contain the non-snRNP splicing factor SC-35. U1 snRNP shows a more widespread nucleoplasmic staining, outside of coiled bodies and "speckled" structures, relative to the other snRNPs. The association of snRNPs with "speckles" is disrupted by heat shock but enhanced when cells are treated with alpha-amanitin.

scholarly journals The organization of spliceosomal components in the nuclei of higher plants

1995 ◽  
Vol 108 (2) ◽  
pp. 509-518 ◽  
Author(s):  
A.F. Beven ◽  
G.G. Simpson ◽  
J.W. Brown ◽  
P.J. Shaw
Keyword(s):  
Cell Cycle ◽  
In Situ Hybridization ◽  
Heat Shock ◽  
Higher Plants ◽  
Specific Protein ◽  
Double Labelling ◽  
Dynamic Changes ◽  
Coiled Bodies ◽  
Immunofluorescence Labelling

To analyze the organization of spliceosomal snRNPs in plant nuclei, we have used both immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B”, and in situ hybridization with anti-sense probes to conserved regions of U1, U2 and U6 snRNAs. The organization comprises a fibrous interchromatin network, which may include both interchromatin fibrils and granules, and very prominent nuclear and nucleolar-associated bodies. Double labelling with an anti-p80 coilin antibody shows that these are coiled bodies. Dynamic changes in the labelling pattern were observed through the cell cycle, and in response to and on recovery from heat shock. The similarity of this organization to that observed in mammalian nuclei is strong evidence that it is fundamental to the processing of pre-mRNA in eucaryotes in general.

scholarly journals Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

2010 ◽  
Vol 192 (7) ◽  
pp. 1999-2005 ◽  
Author(s):  
Amalia Porta ◽  
Annamaria Eletto ◽  
Zsolt Török ◽  
Silvia Franceschelli ◽  
Attila Glatz ◽  
...  
Keyword(s):  
Heat Shock ◽  
Genetic Modification ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Histoplasma Capsulatum ◽  
Genetic Manipulation ◽  
Microbial Pathogens ◽  
Fluid State ◽  
Shock Response ◽  
Serovar Typhimurium

ABSTRACT So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Δ12-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MΦ) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Δ9-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MΦ along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MΦ of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

1995 ◽  
Vol 108 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Starborg ◽  
E. Brundell ◽  
K. Gell ◽  
C. Larsson ◽  
I. White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Metaphase Chromosomes ◽  
Licensing Factor ◽  
Mammalian Homologue ◽  
Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

scholarly journals DjA1 maintains Golgi integrity via interaction with GRASP65

2019 ◽  
Vol 30 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Jie Li ◽  
Danming Tang ◽  
Stephen C. Ireland ◽  
Yanzhuang Wang
Keyword(s):  
Heat Shock ◽  
Membrane Fusion ◽  
Structure Formation ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Golgi Membrane ◽  
Biochemical Methods ◽  
Golgi Fragmentation ◽  
Golgi Structure

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

scholarly journals Interactions of U2 Gene Loci and Their Nuclear Transcripts with Cajal (Coiled) Bodies: Evidence for PreU2 within Cajal Bodies

2000 ◽  
Vol 11 (9) ◽  
pp. 2987-2998 ◽  
Author(s):  
Kelly P. Smith ◽  
Jeanne Bentley Lawrence
Keyword(s):  
Potential Role ◽  
Coiled Body ◽  
Oligonucleotide Probes ◽  
Primary Transcript ◽  
U2 Snrna ◽  
Rna Foci ◽  
Coiled Bodies ◽  
Snrna Gene

The Cajal (coiled) body (CB) is a structure enriched in proteins involved in mRNA, rRNA, and snRNA metabolism. CBs have been shown to interact with specific histone and snRNA gene loci. To examine the potential role of CBs in U2 snRNA metabolism, we used a variety of genomic and oligonucleotide probes to visualize in situ newly synthesized U2 snRNA relative to U2 loci and CBs. Results demonstrate that long spacer sequences between U2 coding repeats are transcribed, supporting other recent evidence that U2 transcription proceeds past the 3′ box. The presence of bright foci of this U2 locus RNA differed between alleles within the same nucleus; however, this did not correlate with the loci's association with a CB. Experiments with specific oligonucleotide probes revealed signal for preU2 RNA within CBs. PreU2 was also detected in the locus-associated RNA foci, whereas sequences 3′ of preU2 were found only in these foci, not in CBs. This suggests that a longer primary transcript is processed before entry into CBs. Although this work shows that direct contact of a U2 locus with a CB is not simply correlated with RNA at that locus, it provides the first evidence of new preU2 transcripts within CBs. We also show that, in contrast to CBs, SMN gems do not associate with U2 gene loci and do not contain preU2. Because other evidence indicates that preU2 is processed in the cytoplasm before assembly into snRNPs, results point to an involvement of CBs in modification or transport of preU2 RNA.

scholarly journals Molecular chaperones and heat shock proteins in atherosclerosis

2012 ◽  
Vol 302 (3) ◽  
pp. H506-H514 ◽  
Author(s):  
Qingbo Xu ◽  
Bernhard Metzler ◽  
Marjan Jahangiri ◽  
Kaushik Mandal
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Molecular Chaperones ◽  
Mammalian Cells ◽  
Vessel Wall ◽  
Protective Role ◽  
Research Field ◽  
Stressed Cells ◽  
Shock Proteins ◽  
Prevention Of Atherosclerosis

In response to stress stimuli, mammalian cells activate an ancient signaling pathway leading to the transient expression of heat shock proteins (HSPs). HSPs are a family of proteins serving as molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. Physiologically, HSPs play a protective role in the homeostasis of the vessel wall but have an impact on immunoinflammatory processes in pathological conditions involved in the development of atherosclerosis. For instance, some members of HSPs have been shown to have immunoregulatory properties and modification of innate and adaptive response to HSPs, and can protect the vessel wall from the disease. On the other hand, a high degree of sequence homology between microbial and mammalian HSPs, due to evolutionary conservation, carries a risk of misdirected autoimmunity against HSPs expressed on the stressed cells of vascular endothelium. Furthermore, HSPs and anti-HSP antibodies have been shown to elicit production of proinflammatory cytokines. Potential therapeutic use of HSP in prevention of atherosclerosis involves achieving optimal balance between protective and immunogenic effects of HSPs and in the progress of research on vaccination. In this review, we update the progress of studies on HSPs and the integrity of the vessel wall, discuss the mechanism by which HSPs exert their role in the disease development, and highlight the potential clinic translation in the research field.

scholarly journals In situ localization of heat-shock proteins and cell death labelling in the salivary gland of acaricide-treated honeybee larvae

Apidologie ◽  
2006 ◽  
Vol 37 (5) ◽  
pp. 507-516 ◽  
Author(s):  
Elaine C.M. Silva-Zacarin ◽  
Ales Gregorc ◽  
Regina L.M. Silva de Moraes
Keyword(s):  
Cell Death ◽  
Salivary Gland ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Shock Proteins
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