Cajal bodies: Evolutionarily conserved nuclear biomolecular condensates with properties unique to plants.

Proper orchestration of the thousands of biochemical processes that are essential to the life of every cell requires highly organized cellular compartmentalization of dedicated microenvironments. There are 2 ways to create this intracellular segregation to optimize cellular function. One way is to create specific organelles, enclosed spaces bounded by lipid membranes that regulate macromolecular flux in and out of the compartment. A second way is via membraneless biomolecular condensates that form due to to liquid-liquid phase separation. Although research on these membraneless condensates has historically been performed using animal and fungal systems, recent studies have explored basic principles governing the assembly, properties, and functions of membraneless compartments in plants. In this review, we discuss how phase separation is involved in a variety of key processes occurring in Cajal bodies (CBs), a type of biomolecular condensate found in nuclei. These processes include RNA metabolism, formation of ribonucleoproteins involved in transcription, RNA splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles of CBs, we discuss unique plant-specific functions of CBs in RNA-based regulatory pathways such as nonsense-mediated mRNA decay, mRNA retention, and RNA silencing. Finally, we summarize recent progress and discuss the functions of CBs in responses to pathogen attacks and abiotic stresses, responses that may be regulated via mechanisms governed by polyADP-ribosylation. Thus, plant CBs are emerging as highly complex and multifunctional biomolecular condensates that are involved in a surprisingly diverse range of molecular mechanisms that we are just beginning to appreciate.

Functional nuclear retention of pre-mRNA involving Cajal bodies during meiotic prophase in European larch (Larix decidua).

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of posttranscriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, for example, during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored nonfully spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

Core spliceosomal Sm proteins as constituents of cytoplasmic mRNPs in plants.

In recent years, research has increasingly focused on the key role of post-transcriptional regulation of messenger ribonucleoprotein (mRNP) function and turnover. As a result of the complexity and dynamic nature of mRNPs, the full composition of a single mRNP complex remains unrevealed and mRNPs are poorly described in plants. Here we identify canonical Sm proteins as part of the cytoplasmic mRNP complex, indicating their function in the post-transcriptional regulation of gene expression in plants. Sm proteins comprise an evolutionarily ancient family of small RNA-binding proteins involved in pre-mRNA splicing. The latest research indicates that Sm could also impact on mRNA at subsequent stages of its life cycle. In this work we show that in the microsporocyte cytoplasm of Larix decidua, the European larch, Sm proteins accumulate within distinct cytoplasmic bodies, also containing polyadenylated RNA. To date, several types of cytoplasmic bodies involved in the post-transcriptional regulation of gene expression have been described, mainly in animal cells. Their role and molecular composition in plants remain less well established, however. A total of 222 mRNA transcripts have been identified as cytoplasmic partners for Sm proteins. The specific colocalization of these mRNAs with Sm proteins within cytoplasmic bodies has been confirmed via microscopic analysis. The results from this work support the hypothesis, that evolutionarily conserved Sm proteins have been adapted to perform a whole repertoire of functions related to the post-transcriptional regulation of gene expression in Eukaryota. This adaptation presumably enabled them to coordinate the interdependent processes of splicing element assembly, mRNA maturation and processing, and mRNA translation regulation, and its degradation.

Different Patterns of mRNA Nuclear Retention during Meiotic Prophase in Larch Microsporocytes.

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.

Spatial regulation of cytoplasmic snRNP assembly at the cellular level.

Small nuclear ribonucleoproteins (snRNPs) play a crucial role in pre-mRNA splicing in all eukaryotic cells. In contrast to the relatively broad knowledge on snRNP assembly within the nucleus, the spatial organization of the cytoplasmic stages of their maturation remains poorly understood. Nevertheless, sparse research indicates that, similar to the nuclear steps, the crucial processes of cytoplasmic snRNP assembly may also be strictly spatially regulated. In European larch microsporocytes, it was determined that the cytoplasmic assembly of snRNPs within a cell might occur in two distinct spatial manners, which depend on the rate of de novo snRNP formation in relation to the steady state of these particles within the nucleus. During periods of moderate expression of splicing elements, the cytoplasmic assembly of snRNPs occurred diffusely throughout the cytoplasm. Increased expression of both Sm proteins and U snRNA triggered the accumulation of these particles within distinct, non-membranous RNP-rich granules, which are referred to as snRNP-rich cytoplasmic bodies.

Molecular evidence that rough endoplasmic reticulum is the site of calreticulin translation in Petunia pollen tubes growing in vitro.

KEY MESSAGE: In germinating pollen grains and growing pollen tubes, CRT is translated on ER membrane-bound ribosomes in the regions where its activity is required for stabilization of tip-focused Ca (2+) gradient. Pollen tube growth requires coordination of signaling, exocytosis, and actin cytoskeletal organization. Many of these processes are thought to be controlled by finely tuned regulation of cytoplasmic Ca(2+) in discrete regions of the tube cytoplasm. Most notably, a mechanism must function to maintain a steep gradient of Ca(2+) that exists at the tip of growing pollen tube. Several pieces of evidence point to calreticulin (CRT) as a key Ca(2+)-binding/-buffering protein involved in pollen germination and pollen tube growth. We previously hypothesized that in germinating pollen and growing tubes, CRT is translated on the ribosomes associated with endoplasmic reticulum (ER) in the regions where its activity might be required. In this report, we have addressed this idea by identifying the sites where CRT mRNA, CRT protein, 18S rRNA, and rough ER are localized in Petunia pollen tubes. We observed all four components in the germinal aperture of pollen grains and in subapical regions of elongating tubes. These results seem to support our idea that CRT is translated on ER membrane-bound ribosomes during pollen germination and pollen tube growth. In elongated pollen tubes, we found CRT mainly localized in the subapical zone, where ER and Golgi stacks are abundant. In eukaryotic cells, these organelles serve as mobile intracellular stores of easily releasable Ca(2+), which can be buffered by proteins such as CRT. Therefore, we postulate that subapical-localized CRT is involved in pollen tube growth by maintaining the stable tip-focused Ca(2+) gradient and thus modulating local Ca(2+) concentration within the tube cytoplasm.

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 (Kolowerzo 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 (Piecinski et al. in Sex Plant Reprod 21:247-257, 2008; Niedojadlo 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.

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 Piecinski 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.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants.

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Some aspects of purinergic signaling in the ventricular system of porcine brain.

BACKGROUND: Numerous signaling pathways function in the brain ventricular system, including the most important - GABAergic, glutaminergic and dopaminergic signaling. Purinergic signalization system - comprising nucleotide receptors, nucleotidases, ATP and adenosine and their degradation products - are also present in the brain. However, the precise role of nucleotide signalling pathway in the ventricular system has been not elucidated so far. The aim of our research was the identification of all three elements of purinergic signaling pathway in the porcine brain ventricular system. RESULTS: Besides nucleotide receptors on the ependymocytes surface, we studied purines and pyrimidines in the CSF, including mechanisms of nucleotide signaling in the swine model (Sus scrofa domestica). The results indicate presence of G proteins coupled P2Y receptors on ependymocytes and also P2X receptors engaged in fast signal transmission. Additionally we found in CSF nucleotides and adenosine in the concentration sufficient to P receptors activation. These extracellular nucleotides are metabolised by adenylate kinase and nucleotidases from at least two families: NTPDases and NPPases. A low activity of these nucleotide metabolising enzymes maintains nucleotides concentration in ventricular system in micromolar range. ATP is degraded into adenosine and inosine. CONCLUSIONS: Our results confirm the thesis about cross-talking between brain and ventricular system functioning in physiological as well as pathological conditions. The close interaction of brain and ventricular system may elicit changes in qualitative and quantitative composition of purines and pyrimidines in CSF. These changes can be dependent on the physiological state of brain, including pathological processes in CNS.

Poly(A) RNA a new component of Cajal bodies.

In European larch microsporocytes, spherical structures 0.5 to 6 microm in diameter are present in which poly(A) RNA accumulates. There were one to several bodies per cell and they were often present in the vicinity of the nucleolus. No nascent transcripts were observed within them. Splicing factors of the SR family, including protein SC35, which participates in bringing the 3' and 5' sites closer in the splicing reaction, were also not observed. The absence of the above-mentioned elements within bodies containing poly(A) RNA disqualifies them as sites of synthesis and preliminary stages of primary transcript maturation. However, they contained abundant elements of the splicing machinery commonly occurring in Cajal bodies, i.e., Sm proteins or small nuclear RNA (snRNA). The molecular composition as well as the characteristic ultrastructure of bodies containing poly(A) RNA proves that these were Cajal bodies. This is the first report of such poly(A) RNA localization.