Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin.

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains.

The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.

Emerging and re-emerging themes in co-transcriptional pre-mRNA splicing.

Proper gene expression requires the collaborative effort of multiple macromolecular machines to produce functional messenger RNA. As RNA polymerase II (RNA Pol II) transcribes DNA, the nascent pre-messenger RNA is heavily modified by other complexes such as 5' capping enzymes, the spliceosome, the cleavage, and polyadenylation machinery as well as RNA-modifying/editing enzymes. Recent evidence has demonstrated that pre-mRNA splicing and 3' end cleavage can occur on similar timescales as transcription and significantly cross-regulate. In this review, we discuss recent advances in co-transcriptional processing and how it contributes to gene regulation. We highlight how emerging areas-including coordinated splicing events, physical interactions between the RNA synthesis and modifying machinery, rapid and delayed splicing, and nuclear organization-impact mRNA isoforms. Coordination among RNA-processing choices yields radically different mRNA and protein products, foreshadowing the likely regulatory importance of co-transcriptional RNA folding and co-transcriptional modifications that have yet to be characterized in detail.

p53 mediates target gene association with nuclear speckles for amplified RNA expression.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages.

In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.

Nuclear speckle integrity and function require TAO2 kinase.

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

PABPN1 aggregation is driven by Ala expansion and poly(A)-RNA binding, leading to CFIm25 sequestration that impairs alternative polyadenylation.

Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.

Trnp1 organizes diverse nuclear membrane-less compartments in neural stem cells.

TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.

Nuclear speckles - a driving force in gene expression.

Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which are splicing factors, that together display complex biophysical properties dictating nuclear speckle formation and maintenance. Although these nuclear bodies were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions in modulation of gene activity. Major advancements in genomic mapping techniques combined with microscopy approaches have enabled insights into the roles nuclear speckles may play in enhancing gene expression, and how gene positioning to specific nuclear landmarks can regulate gene expression and RNA processing. Some studies have drawn a link between nuclear speckles and disease. Certain maladies either involve nuclear speckles directly or dictate the localization and reorganization of many nuclear speckle factors. This is most striking during viral infection, as viruses alter the entire nuclear architecture and highjack host machinery. As discussed in this Review, nuclear speckles represent a fascinating target of study not only to reveal the links between gene positioning, genome subcompartments and gene activity, but also as a potential target for therapeutics.

ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5' splice site motifs within nuclear speckles.

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Using high-throughput sequencing of cellular fractions, we now demonstrate that IPA transcripts require the zinc finger protein ZFC3H1 for their nuclear retention and degradation. Using reporter mRNAs, we demonstrate that ZFC3H1 promotes the nuclear retention of mRNAs with intact 5'SS motifs by sequestering them into nuclear speckles. Furthermore, we find that U1-70K, a component of the spliceosomal U1 snRNP, is also required for the nuclear retention of these reporter mRNAs and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of reporter mRNAs with 5'SS motifs. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs by sequestering them into nuclear speckles.

A systematic analysis of circRNAs in subnuclear compartments.

CircRNAs are an important class of RNAs with diverse cellular functions in human physiology and disease. A thorough knowledge of circRNAs including their biogenesis and subcellular distribution is important to understand their roles in a wide variety of processes. However, the analysis of circRNAs from total RNA sequencing data remains challenging. Therefore, we developed Calcifer, a versatile workflow for circRNA annotation. Using Calcifer, we analysed APEX-Seq data to compare circRNA occurrence between whole cells, nucleus and subnuclear compartments. We generally find that circRNAs show higher abundance in whole cells compared to nuclear samples, consistent with their accumulation in the cytoplasm. The notable exception is the single-exon circRNA circCANX(9), which is unexpectedly enriched in the nucleus. In addition, we observe that circFIRRE prevails over the linear lncRNA FIRRE in both the cytoplasm and the nucleus. Zooming in on the subnuclear compartments, we show that circRNAs are strongly depleted from nuclear speckles, indicating that excess splicing factors in this compartment counteract back-splicing. Our results thereby provide valuable insights into the subnuclear distribution of circRNAs. Regarding circRNA function, we surprisingly find that the majority of all detected circRNAs possess complete open reading frames with potential for cap-independent translation. Overall, we show that Calcifer is an easy-to-use, versatile and sustainable workflow for the annotation of circRNAs which expands the repertoire of circRNA tools and allows to gain new insights into circRNA distribution and function.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Nyamanini Virus Nucleoprotein and Phosphoprotein Organize Viral Inclusion Bodies That Associate with Host Biomolecular Condensates in the Nucleus.

Many mononegaviruses form inclusion bodies (IBs) in infected cells. However, little is known about nuclear IBs formed by mononegaviruses, since only a few lineages of animal-derived mononegaviruses replicate in the nucleus. In this study, we characterized the IBs formed by Nyamanini virus (NYMV), a unique tick-borne mononegavirus undergoing replication in the nucleus. We discovered that NYMV forms IBs, consisting of condensates and puncta of various sizes and morphologies, in the host nucleus. Likewise, we found that the expressions of NYMV nucleoprotein (N) and phosphoprotein (P) alone induce the formation of condensates and puncta in the nucleus, respectively, even though their morphologies are somewhat different from the IBs observed in the actual NYMV-infected cells. In addition, IB-like structures can be reconstructed by co-expressions of NYMV N and P, and localization analyses using a series of truncated mutants of P revealed that the C-terminal 27 amino acid residues of P are important for recruiting P to the condensates formed by N. Furthermore, we found that nuclear speckles, cellular biomolecular condensates, are reorganized and recruited to the IB-like structures formed by the co-expressions of N and P, as well as IBs formed in NYMV-infected cells. These features are unique among mononegaviruses, and our study has contributed to elucidating the replication mechanisms of nuclear-replicating mononegaviruses and the virus-host interactions.

EARLY FLOWERING 3 represses the nighttime growth response to sucrose in Arabidopsis.

Plant growth depends on the supply of carbohydrates produced by photosynthesis. Exogenously applied sucrose promotes the growth of the hypocotyl in Arabidopsis thaliana seedlings grown under short days. Whether this effect of sucrose is stronger under the environmental conditions where the light input for photosynthesis is limiting remains unknown. We characterised the effects of exogenous sucrose on hypocotyl growth rates under light compared to simulated shade, during different portions of the daily cycle. The strongest effects of exogenous sucrose occurred under shade and during the night; i.e., the conditions where there is reduced or no photosynthesis. Conversely, a faster hypocotyl growth rate, predicted to enhance the demand of carbohydrates, did not associate to a stronger sucrose effect. The early flowering 3 (elf3) mutation strongly enhanced the impact of sucrose on hypocotyl growth during the night of a white-light day. This effect occurred under short, but not under long days. The addition of sucrose enhanced the fluorescence intensity of ELF3 nuclear speckles. The elf3 mutant showed increased abundance of PHYTOCHROME INTERACTING FACTOR4 (PIF4), which is a transcription factor required for a full response to sucrose. Sucrose increased PIF4 protein abundance by post-transcriptional mechanisms. Under shade, elf3 showed enhanced daytime and reduced nighttime effects of sucrose. We conclude that ELF3 modifies the responsivity to sucrose according to the time of the daily cycle and the prevailing light or shade conditions.

Speculating on the Roles of Nuclear Speckles: How RNA-Protein Nuclear Assemblies Affect Gene Expression.

Nuclear speckles are eukaryotic nuclear bodies enriched in splicing factors. Their exact purpose has been a matter of debate. The different proposed roles of nuclear speckles are reviewed and an additional layer of function is put forward, suggesting that by accumulating splicing factors within them, nuclear speckles can buffer the nucleoplasmic levels of splicing factors available for splicing and thereby modulate splicing rates. These findings build on the already established model that nuclear speckles function as a storage/recycling site for splicing factors. Many studies have demonstrated proximity between nuclear speckles and sites of active transcription, suggesting that this juxtaposition can enhance the rates of gene expression. It is found that nuclear speckle disassembly increases splicing factor availability in the nucleoplasm, leading to an increase in splicing rates and faster release of nascent transcripts from the gene after transcription. Altogether, this era in which genomic and imaging approaches are applied to study nuclear organization has expanded the outlook on the possible roles of nuclear speckles.

Nuclear speckle fusion via long-range directional motion regulates speckle morphology after transcriptional inhibition.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar long-range speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.

G-Quadruplex Structures Colocalize with Transcription Factories and Nuclear Speckles Surrounded by Acetylated and Dimethylated Histones H3.

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.

Nuclear retention of mRNAs - quality control, gene regulation and human disease.

Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of gene regulation, which enables the cell to rapidly respond to stress, viral infection, differentiation cues or changing environmental conditions. Focusing on mammalian cells, we discuss recent insights into the mechanisms and functions of nuclear retention, describe retention-promoting features in protein-coding transcripts and propose mechanisms for their regulated release into the cytoplasm. Moreover, we discuss examples of how aberrant nuclear retention of mRNAs may lead to human diseases.

Reorganization of the nuclear compartments involved in transcription and RNA processing in myonuclei of type I spinal muscular atrophy.

Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by the loss or mutation of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to the degeneration of motor neurons and muscular atrophy. In this study, we analyzed the nuclear reorganization in human skeletal myofibers from a type I SMA patient carrying a deletion of exons 7 and 8 in the SMN1 gene and two SMN2 gene copies and showing reduced SMN protein levels in the muscle compared with those in control samples. The morphometric analysis of myofiber size revealed the coexistence of atrophic and hypertrophic myofibers in SMA samples. Compared with controls, both nuclear size and the nuclear shape factor were significantly reduced in SMA myonuclei. Nuclear reorganization in SMA myonuclei was characterized by extensive heterochromatinization, the aggregation of splicing factors in large interchromatin granule clusters, and nucleolar alterations with the accumulation of the granular component and a loss of fibrillar center/dense fibrillar component units. These nuclear alterations reflect a severe perturbation of global pre-mRNA transcription and splicing, as well as nucleolar dysfunction, in SMA myofibers. Moreover, the finding of similar nuclear reorganization in both atrophic and hypetrophic myofibers provides additional support that the SMN deficiency in SMA patients may primarily affect the skeletal myofibers.