17 research outputs found
C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon
Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology
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Identification and Characterization of Cytoplasmic Processing Bodies (P Bodies) in Saccharomyces Cerevisiae
An important aspect of regulating gene expression is the interplay between mRNA translation and its degradation. In the work presented here, I, in some cases in collaboration with others, provide insights into how mRNA translation and decay are connected and how they interact with each other to regulate gene expression. This work can be summarized as follows:First, I identified cytoplasmic processing bodies (P bodies) in yeast, which are sites where mRNAs can be decapped and degraded in a 5' to 3' manner. We base our conclusion on three key observations. First, factors involved in the 5' to 3' decay pathway accumulate in P bodies.Second, P bodies change in size when the flux of mRNA decay pathways is perturbed. For example, they decrease in size when entry into decapping is inhibited, and increase in size when decapping is blocked.Third, mRNAs trapped in the process of decay accumulate in P bodies. Second, in a collaborative effort, I have further characterized P bodies. This work involved addressing the role of RNA in P body formation and the relationship of P bodies to translation. Our results suggest that P bodies are dynamic and their size is affected by a range of cellular perturbations. We also provide evidence that P bodies are sensitive to the translational status of the cell and represent sites where translationally repressed mRNAs accumulate, and where they can be subjected to 5' to 3' decay. Third, I have determined that Nonsense-mediated decay (NMD), a quality control mechanism that rapidly degrades aberrant mRNAs, involves targeting of aberrant mRNAs to P bodies. In addition, I have identified specific roles for Upf proteins in the process of NMD: Upf1p is involved in targeting mRNAs to P bodies and Upf2p and Upf3p playing a role in degradation of the aberrant mRNAs within P bodies.The identification of P bodies has direct implications on regulation of mRNA decapping, of both normal and aberrant mRNAs. The similarities of P bodies with mRNA storage granules in other organisms imply that P bodies will play a major role in regulation of translationally repressed mRNAs
Processing bodies require RNA for assembly and contain nontranslating mRNAs
Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation
Capsids accumulate in the mid-pachytene gonad core as adults age at 15°C.
<p>(A–D) Longitudinal, optical sections through gonads showing increased accumulation of capsids (green) with adult age, as indicated. The gonad in panel B is immunostained for HIM-4/hemicentin (red) to visualize the apical membrane (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1B</a>). Note that capsids localize predominately in the core, outside the ring channel and away from nuclei (blue, DAPI). (C) Capsids grouped in wavy lines and tangles of lines in the mid-pachytene region of a day 3 adult. (D) Low magnification of the mid-pachytene region. Note variation in capsid abundance between the two sides of the gonad core (double-headed arrow), and that the wavy lines of capsids disappear as germ cells move proximally into late pachytene (bracketed region). (E–G) The late-pachytene/diplotene region, stained and imaged as for panel B; two of the somatic sheath cells that surround the gonad are visible in this image. Germ cells from regions F and G are shown at high magnification, oriented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1B</a>. An optical rotation of a similar region of the gonad is shown in Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591.s005" target="_blank">Video S1</a>. (H) Optical section through germ nuclei in the late-pachytene/diplotene region showing capsids (green) and P granules (red, αPGL-1). Note that capsids localize close to the nuclear envelope, but most are not directly on, or within, P granules. Electron micrographs of capsids within P granules are shown in Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591.s001" target="_blank">Figure S1H</a>. Scale bars: A–C, F–H (5 µm), D–E (10 µm).</p
A subset of microtubules in the mid-pachytene region are inhibitor-resistant, or stable.
<p>(A) Optical section through the superapical plane of an untreated day 1 adult gonad showing the dense network of long microtubules (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1A</a> for section orientation). The inset shows a high magnification of the dashed box, with an arrowhead marking the end of a single microtubule traced from the indicated germ cell (asterisk). (B,C) Optical sections through the central planes of untreated, day 1 adult gonads; the gonads are immunostained for both microtubules and <i>Cer1</i> capsids. Note that capsids are concentrated in linear or irregular shapes, while the microtubules are distributed uniformly. (D) Optical section through the superapical plane of a day 1 adult gonad treated with nocodazole; the bracketed region is the same late-pachytene/diplotene region, and the same optical plane, as for the untreated gonad in panel A. Note that most microtubules have disappeared from this region after nocodazole treatment, in contrast to the numerous “stable” microtubules that remain in the mid-pachytene region. (E,F) Microtubules in the distal, mitotic regions of gonads before (E) and after (F) nocodazole treatment. The arrow in panel E indicates an example of a mitotic spindle. Most of the brightest spots of tubulin visible in panel F co-localize with centrosomes (data not shown). (G) Optical section through the central plane of a nocodazole-treated gonad. This sectional view combined with that in panel D illustrates that most of the stable microtubules in the mid-pachytene region are in the superapical zone of the core (arrowheads). Note that most of the microtubules in the late-pachytene/diplotene germ cells have depolymerized; stable microtubules that appear at the periphery of the gonad (arrow) are outside of germ cells and within the thin cell bodies of somatic sheath cells. (H) Stable microtubules in nocodazole-treated oocytes; the oocytes advance in age right to left (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1A</a>). Note the focus of stable microtubules (arrow) near the oocyte nucleus, and the abrupt disappearance of all stable microtubules in the more mature oocyte to the left; compare with oocyte progression panel D, where numbers indicate oocyte position relative to ovulation. Scale bars: A–C (5 µm), D–H (10 µm).</p
<i>Cer1</i> GAG particles in <i>C. elegans</i> wild strains.
a<p>Data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Palopoli1" target="_blank">[31]</a>.</p
Electron microscopy of VLPs in <i>Caenorhabditis</i>.
<p>Electron microscopy of VLPs in <i>Caenorhabditis</i>.</p
Nematode gonads contain VLPs.
<p>(A) Diagrams of longitudinal and cross sections through one arm of the adult hermaphrodite gonad with germ nuclei indicated in dark blue; somatic sheath cells that surround the gonad are not shown. Optical sectioning planes referred to in this paper are indicated in the cross-section (dashed lines). During development, germ cells move from the distal (mitotic) end of the gonad toward the proximal end, where they differentiate as oocytes. (B) Enlarged diagram of two germ cells with ring channels opening to the gonad core. Basket and core microtubules (MTs) are drawn in red. (C) Linear representation of germ cell development. Red shading indicates region-specific conditions in germ cells that might impede viral replication. These include the state of the nuclear envelope <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-McCarter1" target="_blank">[67]</a>, P granules <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Strome1" target="_blank">[21]</a>, transcription <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Walker1" target="_blank">[68]</a>, chromatin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Greenstein1" target="_blank">[69]</a>, and cytoplasmic flow <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Wolke1" target="_blank">[27]</a>; ooc = oocytes, emb = embryos. (D–E) Electron micrographs of <i>C. japonica</i> VLPs. Low magnification in panel E shows a small cluster of VLPs (arrow) on top of a P granule; arrowheads indicate examples of nuclear pores. See also Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591.s001" target="_blank">Figure S1A, S1B, S1C</a>). (F) <i>C. elegans</i> VLPs showing variation in internal electron density; note curved, rod-like bodies within the VLPs in the middle panel. Scale bars: D (0.2 µm), E (0.5 µm), F (0.1 µm).</p