Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein

Vetter, Guillaume; Hily, Jean-Michel; Klein, Elodie; Schmidlin, Laure; Haas, Muriel; Merkle, Thomas; Gilmer, David

doi:10.1099/vir.0.80142-0

Cited by 53 publications

(46 citation statements)

References 43 publications

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“…-GFP from plasmid pGII⍀MP⌬C55:GFP and Man-1-mRFP from pBP30-GmMan1:RFP was achieved by biolistic bombardment as described previously (101). BY-2 suspension culture cells were fixed and immunostained according to the method de-scribed in reference 87, except that cell wall digestion was performed for 10 min and that mouse monoclonal anti-␣-tubulin (clone DM1B, dilution 1:1,000) was used as a primary antibody.…”

Section: ⌬C55mentioning

confidence: 99%

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Ashby

Boutant

Seemanpillai

et al. 2006

J Virol

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The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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Section: ⌬C55mentioning

confidence: 99%

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Ashby

Boutant

Seemanpillai

et al. 2006

J Virol

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“…Full-length cDNAs of AtRRP6L1, -2, and -3 were obtained by reverse transcriptase (RT) PCR and fused to enhanced green fluorescent protein (EGFP) in N-and C-terminal orientations in pBinH, a pBin-PLUS derivative (48) containing the cauliflower mosaic virus 35S promoter and terminator sequences and a hygromycin resistance cassette. Biolistic transformation of tobacco BY2 cells and analysis by confocal microscopy were as described previously (49). For stable transformants, Arabidopsis plants were transformed by the floral-dip method (13).…”

Section: Methodsmentioning

confidence: 99%

Degradation of a Polyadenylated rRNA Maturation By-Product Involves One of the Three RRP6-Like Proteins in Arabidopsis thaliana

Lange

Holec

Cognat

et al. 2008

Molecular and Cellular Biology

117

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Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6⌬ strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5 part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.The exoribonucleolytic activity of the exosome is fundamental to many aspects of RNA metabolism. The exosome is involved in mRNA turnover and degradation of defective and cryptic transcripts, but also in processing of the 3Ј extremities of a variety of noncoding RNAs and elimination of RISCcleaved mRNA (for recent reviews, see references 27 and 46). While most exosome functions were initially characterized in the yeast Saccharomyces cerevisiae, related complexes are present in all higher eukaryotes investigated and in several Archaea (4, 9-11, 17, 26, 29, 35, 36).Eukaryotic exosomes are composed of a core complex with which cytoplasm-and nucleus-specific subunits associate (reviewed in reference 38). One of these subunits, Rrp6p, a member of the RNase D family, associates with the nuclear exosome in yeast (2). Its human counterpart, PM/Scl-100, is predominantly nuclear but is also detected in the cytoplasm (7). Rrp6p plays a role in nuclear mRNA surveillance and in the degradation of rRNA maturation by-products or intergenic transcripts (30,33,47,51). In addition, Rrp6p is involved in the final steps in processing several noncoding RNAs (1, 6).In yeast, the TRAMP complex polyadenylates RNA substrates, which triggers their degradation by the nuclear exosome (30,47,51). In higher eukaryotes, evidence for polyadenylation of nuclear transcripts destined for degradation is emerging. Short poly(A) tails were detected upon cotranscriptional cleavage of human ␤-globin and murine serum albumin pre-mRNA (50). Human rRNA can also be polyadenylated at putative sites of endonucleolytic cleavage (44). In plants, polyadenylation of nuclear noncoding RNA also occurs, as polyadenylated transcripts of the low-abundance 5S rRNA spacer were reported in Nicotiana (20). During revision of our manuscript, a genome-wide search for exosome substrates revealed that a wide range of nuclear noncoding transcripts a...

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“…For GmManI-RFP, the same procedure was employed except that the PCR-amplified RFP was subcloned into the BP30 vector of Nebenfü hr et al (1999) to get pBP30-GmManI:RFP. The LeSec13:GFP-expressing cell line was transfected by biolistics using the procedure described by Vetter et al (2004). Cells were observed between 18 and 24 h post-transfection except for BiP:DsRed, which was visualized between 36 and 48 h post-transfection.…”

Section: Preparation Of Rfp Constructs and Biolistic Transformationmentioning

confidence: 99%

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

et al. 2005

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Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPIIcoat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.

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Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein

Cited by 53 publications

References 43 publications

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Degradation of a Polyadenylated rRNA Maturation By-Product Involves One of the Three RRP6-Like Proteins in Arabidopsis thaliana

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

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