RNA is required for the integrity of multiple nuclear and cytoplasmic membrane‐less RNP granules

Numerous membrane‐less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super‐enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane‐less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.     

The nuclear bodies formed by histone demethylase KDM7A

Dear Editor, In the nucleus of higher eukaryotes, chromatin occupies only a small proportion of the nuclear space, while many proteins and RNAs segregate into membrane-less nuclear bodies (NBs).These NBs follow a stochastic or ordered assembly model and constantly exchange components with the surrounding nucleoplasm (Jain et al., 2016).Typical NBs include nucleoli, nuclear speckles, paraspeckles, PML bodies, Cajal bodies, polycomb bodies and Sam68 bodies,which play critical roles in various biological processes such as ribosome assembly, RNA processing, and protein modification.The dysfunction of nuclear bodies may cause diseases, such as cancer (Li et al., 2019).     

All small nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP localize to nucleoli; Identification of the nucleolar localization element of U6 snRNA

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3' end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3'-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3' hydroxyl of U6 snRNA to a 3' phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies.     

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

Background

The biogenesis of spliceosomal snRNPs takes place in both the cytoplasm where Sm core proteins are added and snRNAs are modified at the 5′ and 3′ termini and in the nucleus where snRNP-specific proteins associate. U1 snRNP consists of U1 snRNA, seven Sm proteins and three snRNP-specific proteins, U1-70K, U1A, and U1C. It has been shown previously that after import to the nucleus U2 and U4/U6 snRNP-specific proteins first appear in Cajal bodies (CB) and then in splicing speckles. In addition, in cells grown under normal conditions U2, U4, U5, and U6 snRNAs/snRNPs are abundant in CBs. Therefore, it has been proposed that the final assembly of these spliceosomal snRNPs takes place in this nuclear compartment. In contrast, U1 snRNA in both animal and plant cells has rarely been found in this nuclear compartment.

Methodology/Principal Findings

Here, we analysed the subnuclear distribution of Arabidopsis U1 snRNP-specific proteins fused to GFP or mRFP in transiently transformed Arabidopsis protoplasts. Irrespective of the tag used, U1-70K was exclusively found in the nucleus, whereas U1A and U1C were equally distributed between the nucleus and the cytoplasm. In the nucleus all three proteins localised to CBs and nucleoli although to different extent. Interestingly, we also found that the appearance of the three proteins in nuclear speckles differ significantly. U1-70K was mostly found in speckles whereas U1A and U1C in ∼90% of cells showed diffuse nucleoplasmic in combination with CBs and nucleolar localisation.

Conclusions/Significance

Our data indicate that CBs and nucleolus are involved in the maturation of U1 snRNP. Differences in nuclear accumulation and distribution between U1-70K and U1A and U1C proteins may indicate that either U1-70K or U1A and U1C associate with, or is/are involved, in other nuclear processes apart from pre-mRNA splicing.     

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway.

BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.     

In vivo analysis of Cajal body movement, separation, and joining in live human cells

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.     

Nuclear hubs built on RNAs and clustered organization of the genome

RNAs play diverse roles in formation and function of subnuclear compartments, most of which are associated with active genes. NEAT1 and NEAT2/MALAT1 exemplify long non-coding RNAs (lncRNAs) known to function in nuclear bodies; however, we suggest that RNA biogenesis itself may underpin much nuclear compartmentalization. Recent studies show that active genes cluster with nuclear speckles on a genome-wide scale, significantly advancing earlier cytological evidence that speckles (aka SC-35 domains) are hubs of concentrated pre-mRNA metabolism. We propose the ‘karyotype to hub’ hypothesis to explain this organization: clustering of genes in the human karyotype may have evolved to facilitate the formation of efficient nuclear hubs, driven in part by the propensity of ribonucleoproteins (RNPs) to form large-scale condensates. The special capacity of highly repetitive RNAs to impact architecture is highlighted by recent findings that human satellite II RNA sequesters factors into abnormal nuclear bodies in disease, potentially co-opting a normal developmental mechanism.     

The nucleolus: a site of ribonucleoprotein maturation

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.     

Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.