Interplay of cGAS with micronuclei: Regulation and diseases.

In higher eukaryotes, sophisticate regulation of genome function requires all chromosomes to be packed into a single nucleus. Micronucleus (MN), the dissociative nucleus-like structure frequently observed in aging and multiple disease settings, has critical, yet under-recognized, pathophysiological functions. Micronuclei (MNi) have recently emerged as major sources of cytosolic DNA that can activate the cGAS-STING axis in a cell-intrinsic manner. However, MNi induced from different genotoxic stressors display great heterogeneity in binding or activating cGAS and the signaling responses downstream of the MN-induced cGAS-STING axis have divergent outcomes including autoimmunity, autoinflammation, metastasis, or cell death. Thus, full characterization of molecular network underpinning the interplay of cGAS and MN is important to elucidate the pathophysiological roles of immunogenic MN and design improved drugs that selectively target cancer via boosting the MN-derived cGAS-STING axis. Here, we summarize our current understanding of the mechanisms for self-DNA discrimination by cGAS. We focus on discussing how MN immunogencity is dictated by multiple mechanisms including integrity of micronuclear envelope, state of nucleosome and DNA, competitive factors, damaged mitochondrial DNA and micronucleophagy. We also describe emerging links between immunogenic MN and human diseases including cancer, neurodegenerative diseases and COVID-19. Particularly, we explore the exciting concept of inducing immunogenic MN as a therapeutic approach in treating cancer. We propose a new theoretical framework to describe immunogenic MN as a biological sensor to modulate cellular processes in response to genotoxic stress and provide perspectives on developing novel experimental approaches to unravel the complexity of MN immunogenicity regulation and immunogenic MN pathophysiology.

RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination.

RAG1 and RAG2 form a tetramer nuclease to initiate V(D)J recombination in developing T and B lymphocytes. The RAG1 protein evolves from a transposon ancestor and possesses nuclease activity that requires interaction with RAG2. Here, we show that the human RAG1 aggregates in the nucleus in the absence of RAG2, exhibiting an extremely low V(D)J recombination activity. In contrast, RAG2 does not aggregate by itself, but it interacts with RAG1 to disrupt RAG1 aggregates and thereby activate robust V(D)J recombination. Moreover, RAG2 from mouse and zebrafish could not disrupt the aggregation of human RAG1 as efficiently as human RAG2 did, indicating a species-specific regulatory mechanism for RAG1 by RAG2. Therefore, we propose that RAG2 coevolves with RAG1 to release inert RAG1 from aggregates and thereby activate V(D)J recombination to generate diverse antigen receptors in lymphocytes.

Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes.

The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

Alteration of SC35 localization by transfection reagents.

Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Ever since the discovery of transfection many side effects or artifacts caused by transfection reagents have been reported. Here, we show that the transfection reagent, JetPRIME alters the localization of the splicing protein SC35 widely used as a nuclear speckle marker. We demonstrate that transfection of plasmids with JetPRIME leads to enlarged SC35 speckles and SC35 cytoplasmic granules. By contrast, transfection of the same plasmid with Lipofectamine 3000 does not have any effect on SC35 localization. The formation of SC35 cytoplasmic granules by JetPRIME-mediated transfection is independent of exogenous expression by plasmid and although similar in morphology they are distinct from P-bodies and stress granules. This method of transfection affected only SC35 and phosphorylated SR proteins but not the nuclear speckles. The JetPRIME-mediated transfection also showed compromised transcription in cells with enlarged SC35 speckles. Our work indicates that the use of JetPRIME alters SC35 localization and can affect gene expression and alternative splicing. Therefore, caution should be exercised when interpreting results after the use of a transient transfection system, particularly when the subject of the study is the function of a protein in the control of gene expression or mRNA splicing.

Biomolecular condensates in neurodegeneration and cancer.

The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane-less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house-keeping, stress-response and cell type-specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.

53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage.

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.

Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing.

Proteins and RNAs inside the cell nucleus are organized into distinct phases, also known as liquid-liquid phase separated (LLPS) droplet organelles or nuclear bodies. These regions exist within the spaces between chromatin-rich regions but their function is tightly linked to gene activity. They include major microscopically-observable structures such as the nucleolus, paraspeckle and Cajal body. The biochemical and assembly factors enriched inside these microenvironments regulate chromatin structure, transcription, and RNA processing, and other important cellular functions. Here, we describe published evidence that suggests nuclear bodies are bona fide LLPS droplet organelles and major regulators of the processes listed above. We also outline an updated "Supply or Sequester" model to describe nuclear body function, in which proteins or RNAs are supplied to surrounding genomic regions or sequestered away from their sites of activity. Finally, we describe recent evidence that suggests these microenvironments are both reflective and drivers of diverse pathophysiological states.

The long noncoding RNA NEAT1 and nuclear paraspeckles are up-regulated by the transcription factor HSF1 in the heat shock response.

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

C3G dynamically associates with nuclear speckles and regulates mRNA splicing.

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments.

Nuclear domain 10 (ND10) bodies are small (0.1-1 µM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-ß. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-ß in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-ß and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.

DNA loop domain organization in nucleoids from cells of different types.

The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type.

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

Nuclear speckles are detention centers for transcripts containing expanded CAG repeats.

The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular "toxicity" of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures.

Empirical Derivation of Correction Factors for Human Spiral Ganglion Cell Nucleus and Nucleolus Count Units.

OBJECTIVE: Profile count method for estimating cell number in sectioned tissue applies a correction factor for double count (resulting from transection during sectioning) of count units selected to represent the cell. For human spiral ganglion cell counts, we attempted to address apparent confusion between published correction factors for nucleus and nucleolus count units that are identical despite the role of count unit diameter in a commonly used correction factor formula. STUDY DESIGN: We examined a portion of human cochlea to empirically derive correction factors for the 2 count units, using 3-dimensional reconstruction software to identify double counts. SETTING: The Neurotology and House Histological Temporal Bone Laboratory at University of California at Los Angeles. SUBJECTS AND METHODS: Using a fully sectioned and stained human temporal bone, we identified and generated digital images of sections of the modiolar region of the lower first turn of cochlea, identified count units with a light microscope, labeled them on corresponding digital sections, and used 3-dimensional reconstruction software to identify double-counted count units. RESULTS: For 25 consecutive sections, we determined that double-count correction factors for nucleus count unit (0.91) and nucleolus count unit (0.92) matched the published factors. We discovered that nuclei and, therefore, spiral ganglion cells were undercounted by 6.3% when using nucleolus count units. CONCLUSION: We determined that correction factors for count units must include an element for undercounting spiral ganglion cells as well as the double-count element. We recommend a correction factor of 0.91 for the nucleus count unit and 0.98 for the nucleolus count unit when using 20-µm sections.

Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles.

Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.

Identification of three redundant segments responsible for herpes simplex virus 1 ICP0 to fuse with ND10 nuclear bodies.

UNLABELLED: Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a key regulator in both lytic and latent infections. In lytic infection, an important early event is the colocalization of ICP0 to nuclear domain 10 (ND10), the discrete nuclear bodies that impose restrictions on viral expression. ICP0 contains an E3 ubiquitin ligase that degrades promyelocytic leukemia protein (PML) and Sp100, two major components of ND10, and disperses ND10 to alleviate repression. We previously reported that the association between ICP0 and ND10 is a dynamic process that includes three steps: adhesion, fusion, and retention. ICP0 residues 245 to 474, defined as ND10 entry signal (ND10-ES), is a region required for the fusion step. Without ND10-ES, ICP0 adheres at the ND10 surface but fails to enter. In the present study, we focus on characterizing ND10-ES. Here we report the following. (i) Fusion of ICP0 with ND10 relies on specific sequences located within ND10-ES. Replacement of ND10-ES by the corresponding region from ORF61 of varicella-zoster virus did not rescue ND10 fusion. (ii) Three tandem ND10 fusion segments (ND10-FS1, ND10-FS2, and ND10-FS3), encompassing 200 amino acids within ND10-ES, redundantly facilitate fusion. Each of the three segments is sufficient to independently drive the fusion process, but none of the segments by themselves are necessary for ND10 fusion. Only when all three segments are deleted is fusion blocked. (iii) The SUMO interaction motif located within ND10-FS2 is not required for ND10 fusion but is required for the complete degradation of PML, suggesting that PML degradation and ND10 fusion are regulated by different molecular mechanisms. IMPORTANCE: ND10 nuclear bodies are part of the cell-intrinsic antiviral defenses that restrict viral gene expression upon virus infection. As a countermeasure, infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) localizes to ND10s, degrades the ND10 organizer, and disperses ND10 components in order to alleviate repression. We studied the ICP0-ND10 association to delineate elements important for this dynamic interaction and to understand its role in viral replication and host defense. In this work, we show that ICP0 contains three redundant segments to ensure an effective mergence of ICP0 with ND10 nuclear bodies. This is the first study to systematically investigate ICP0 elements that are important for ICP0-ND10 fusion.

Diverse functions and mechanisms of mammalian long noncoding RNAs.

Long noncoding RNAs are becoming increasingly appreciated as major players in gene regulation. They have been reported to play diverse roles in many biological processes. Here, we discuss their discovery, features, and known functions in cells. While not comprehensive, this chapter should serve to illustrate the power and promise of studying long noncoding RNAs.

Construction of synthetic nucleoli and what it tells us about propagation of sub-nuclear domains through cell division.

The cell nucleus is functionally compartmentalized into numerous membraneless and dynamic, yet defined, bodies. The cell cycle inheritance of these nuclear bodies (NBs) is poorly understood at the molecular level. In higher eukaryotes, their propagation is challenged by cell division through an "open" mitosis, where the nuclear envelope disassembles along with most NBs. A deeper understanding of the mechanisms involved can be achieved using the engineering principles of synthetic biology to construct artificial NBs. Successful biogenesis of such synthetic NBs demonstrates knowledge of the basic mechanisms involved. Application of this approach to the nucleolus, a paradigm of nuclear organization, has highlighted a key role for mitotic bookmarking in the cell cycle propagation of NBs.

Alanine repeats influence protein localization in splicing speckles and paraspeckles.

Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.