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.

Dynamic distribution of ARGONAUTE1 (AGO1) and ARGONAUTE4 (AGO4) in Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro.

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth.

Transcriptional activity in diplotene larch microsporocytes, with emphasis on the diffuse stage.

Manuscript provides insights into the biology of long-lived plants, different from Arabidopsis, tomato or grass species that are widely studied. In the European larch the diplotene stage lasts approximately 5 months and it is possible to divide it into several substages and to observe each of them in details. The diplotene stage is a period of intensive microsporocyte growth associated with the synthesis and accumulation of different RNA and proteins. Larch microsporocytes display changes in chromatin morphology during this stage, alternating between 4 short stages of chromatin condensation (contraction) and 5 longer diffusion (relaxation) stages. The occurrence of a diplotene diffusion stage has been observed in many plant species. Interestingly, they have also been observed during spermiogenesis and oogenesis in animals. The aim of this study was to examine whether chromatin relaxation during the diplotene is accompanied by the synthesis and maturation of mRNA. The results reveal a correlation between the diffusion and chromatin decondensation, transcriptional activity. We also found decreasing amount of poly(A) mRNA synthesis in the consecutive diffusion stages. During the early diffusion stages, mRNA is intensively synthesized. In the nuclei large amounts of RNA polymerase II, and high levels of snRNPs were observed. In the late diffusion stages, the synthesized mRNA is not directly subjected to translation but it is stored in the nucleus, and later transported to the cytoplasm and translated. In the last diffusion stage, the level of poly(A) RNA is low, but that of splicing factors is still high. It appears that the mRNA synthesized in early stages is used during the diplotene stage and is not transmitted to dyad and tetrads. In contrast, splicing factors accumulate and are most likely transmitted to the dyad and tetrads, where they are used after the resumption of intense transcription. Similar meiotic process were observed during oogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of chromatin-based regulation of gene expression during meiotic prophase I.