Periodic expression of Sm proteins parallels formation of nuclear Cajal bodies and cytoplasmic snRNP-rich bodies

Small nuclear ribonucleoproteins (snRNPs) play a fundamental role in pre-mRNA processing in the nucleus. The biogenesis of snRNPs involves a sequence of events that occurs in both the nucleus and cytoplasm. Despite the wealth of biochemical information about the cytoplasmic assembly of snRNPs, little is known about the spatial organization of snRNPs in the cytoplasm. In the cytoplasm of larch microsporocytes, a cyclic appearance of bodies containing small nuclear RNA (snRNA) and Sm proteins was observed during anther meiosis. We observed a correlation between the occurrence of cytoplasmic snRNP bodies, the levels of Sm proteins, and the dynamic formation of Cajal bodies. Larch microsporocytes were used for these studies. This model is characterized by natural fluctuations in the level of RNA metabolism, in which periods of high transcriptional activity are separated from periods of low transcriptional activity. In designing experiments, the authors considered the differences between the nuclear and cytoplasmic phases of snRNP maturation and generated a hypothesis about the direct participation of Sm proteins in a molecular switch triggering the formation of Cajal bodies

Transcriptional activity of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of incorporated 5′-bromouracil, the total RNA polymerase II pool and the hypo- (initiation) and hyperphosphorylated (elongation) forms of RNA Pol II. The first stage, which lasts from the multinuclear stage to cellularization, is a period of high transcriptional activity, probably related to the maturation of female gametophyte cells. The second stage, encompassing the period of embryo sac maturity and the progamic phase, involves the transcriptional silencing of cells that will soon undergo fusion with male gametes. During this period in the hyacinth egg cell, there are almost no newly formed transcripts, and only a small pool of RNA Pol II is present in the nucleus. The transcriptional activity of the central cell is only slightly higher than that observed in the egg cell. The post-fertilization stage is related to the transcriptional activation of the zygote and the primary endosperm cell. The rapid increase in the pool of newly formed transcripts in these cells is accompanied by an increase in the pool of RNA Pol II, and the pattern of enzyme distribution in the zygote nucleus is similar to that observed in the somatic cells of the ovule. Our data, together with the earlier results of Pięciński et al. (2008), indicate post-fertilization synthesis and the maturation of numerous mRNA transcripts, suggesting that fertilization in H. orientalis induces the activation of the zygote and endosperm genomes

mRNA accumulation in the Cajal bodies of the diplotene larch microsporocyte

In microsporocytes of the European larch, we demonstrated the presence of several mRNAs in spherical nuclear bodies. In the nuclei of microsporocytes, we observed up to 12 bodies, ranging from 0.5 to 6 μm in diameter, during the prophase of the first meiotic division. Our previous studies revealed the presence of polyadenylated RNA (poly(A) RNA) in these bodies, but did not confirm the presence of nascent transcripts or splicing factors of the SR family. The lack of these molecules precludes the bodies from being the sites of synthesis and early maturation of primary transcripts (Kołowerzo et al., Protoplasma 236:13–19, 2009). However, the bodies serve as sites for the accumulation of splicing machinery, including the Sm proteins and small nuclear RNAs. Characteristic ultrastructures and the molecular composition of the nuclear bodies, which contain poly(A) RNA, are indicative of Cajal bodies (CBs). Here, we demonstrated the presence of several housekeeping gene transcripts—α-tubulin, pectin methylesterase, peroxidase and catalase, ATPase, and inositol-3-phosphate synthase—in CBs. Additionally, we observed transcripts of the RNA polymerase II subunits RPB2 and RPB10 RNA pol II and the core spliceosome proteins mRNA SmD1, SmD2, and SmE. The co-localization of nascent transcripts and mRNAs indicates that mRNA accumulation/storage, particularly in CBs, occurs in the nucleus of microsporocytes

Ribosomal RNA of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

The nucleolar activity of Hyacinthus orientalis L. embryo sac cells was investigated. The distributions of nascent pre-rRNA (ITS1), 26S rRNA and of the 5S rRNA and U3 snoRNA were determined using fluorescence in situ hybridization (FISH). Our results indicated the different rRNA metabolism of the H. orientalis female gametophyte cells before and after fertilization. In the target cells for the male gamete, i.e., the egg cell and the central cell whose activity is silenced in the mature embryo sac (Pięciński et al. in Sex Plant Reprod 21:247–257, 2008; Niedojadło et al. in Planta doi:10.1007/s00425-012-1599-9, 2011), rRNA metabolism is directed at the accumulation of rRNPs in the cytoplasm and immature transcripts in the nucleolus. In both cells, fertilization initiates the maturation of the maternal pre-rRNA and the expression of zygotic rDNA. The resumption of rRNA transcription observed in the hyacinth zygote indicates that in plants, there is a different mechanism for the regulation of RNA Pol I activity than in animals. In synergids and antipodal cells, which have somatic functions, the nucleolar activity is correlated with the metabolic activity of these cells and changes in successive stages of embryo sac development

Poly(A) RNAs including coding proteins RNAs occur in plant Cajal bodies.

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention

Analysis of the fluorescence intensity resulting from <i>in situ</i> hybridisation of poly(A) RNA in the meristematic cells of lupines roots.

<p>There were significant differences in intensity (p<0.05) between the cytoplasm (MC), nucleus (MN) and nuclear bodies (MNB).</p

Localisation of poly(A) RNA in control lupine root cells (A) and after submersion to tap water for 3 h (B).

<p>Arrows indicate CBs, and arrow heads indicate cytoplasmic granules. Bar, 5 µm. Nu- nucleolus. Quantitative analysis of the fluorescence intensity associated with the localisation of poly(A) RNA. Significant differences in the signal intensity in the nuclei and CBs between control and hypoxia-treated cells (C) MNu- control nucleus, MCB control Cajal bodies, HypNu and HypCB- nucleus and Cajal bodies after hypoxia treatment, respectively (p = 0.05).</p

Double labelling of poly(A) RNA and the PANA antigen in the chromocentric (<i>Lupinus</i>) (A–C) and reticular (<i>Allium</i>) (D–F) nuclei of root cells.

<p>In the nucleoplasm of both species, poly(A) RNA is present in nuclear structures (arrows) and does not colocalise with speckles. Representative examples of Pearson correlation coefficients for weak and non-colocalisation of poly(A) mRNA with the PANA antigen in <i>Lupinus luteus</i> and <i>Allium cepa</i> cells (G). A scale bar representing 2 µm is shown. The percentages of weak and non-colocalisation of poly(A) RNA-rich bodies with the PANA antigen are indicated by the Pearson correlation coefficient (H). Error bars represent standard error. Double labelling of poly(A) RNA and U2 snRNA in <i>Lupinus</i> (I–K) and <i>Allium</i> (L–N) cells. Accumulation of poly(A) RNA in nuclear bodies rich in U2 snRNA (arrows). Bar, 5 µm. N-nucleus, Nu- nucleolus</p

Double labelling of a mixture of four mRNAs (A, C) and distinct genes: cyclin B1 mRNA (D, F), peroxidase mRNA (G, I), cytokinin-specific binding protein mRNA (J, L) and pectin methylesterase mRNA (M, O), with Sm proteins (B, E, H, K, N) in <i>Lupinus</i> cells.

<p>The arrows indicate colocalisation of mRNA transcripts with CBs. A stronger signal was observed in the cytoplasm than in the nuclei after <i>in situ</i> hybridisation with the mixture of probes (A, C) and with a probe against cytokinin-specific binding protein mRNA (J, L). % indicates the percentage of nuclei showing the representative immunolocalisation pattern. Bar, 5 µm. N-nucleus, Nu- nucleolus, C- cytoplasm.</p

Double labelling of poly(A) RNA (A) and the elongation form of RNA polymerase II (B) in <i>Lupinu</i> root cells.

<p>Cajal body (arrow) rich in poly(A) RNA near the nucleolus do not colocalise with RNA polymerase II. Double labelling of a newly formed transcript (E, H) and poly(A) RNA (D, G) in lupine cells. If the transcript does not leave the nucleus, no signal occurs in the CB (arrow) (D–F). In cells in which the newly formed RNA is transported to the cytoplasm, a weak signal is observed in the Cajal body (arrow) (G–I). Bar, 5 µm. Fragment of the nucleus from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111780#pone-0111780-g002" target="_blank">Fig 2I</a> (J). BrU-containing RNA localises at the periphery and in small spots in the middle of CB (J). Bar, 1 µm. The percentage of Cajal bodies rich in poly(A) RNA that colocalise with BrU-containing RNA (K, L). The data for experimental treatments and control were conducted, analyzed and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111780#pone-0111780-g002" target="_blank">Figure 2G–H</a>. A scale bar representing 2 µm is shown. Error bars represent standard error. N- nucleus, Nu- nucleolus, C- cytoplasm.</p