Coilin is a protein localized in the nucleus, where it plays a role in the assembly of the Cajal Body and is involved in ribonucleoprotein biogenesis. Our recent research has uncovered new roles for coilin, including its involvement in producing microRNAs and in modifying other proteins through phosphorylation and SUMOylation. We also proposed that coilin could respond to stress signals. In plants, coilin has been shown to help regulate immune genes and activate defense mechanisms, especially in response to stress. In this study, we used two vertebrate models to study coilin function: a human primary foreskin fibroblast cell line deficient in coilin through RNA interference and a newly created zebrafish line with a mutation in the coilin gene generated by CRISPR-Cas9. Transcriptomic analysis in these two models of coilin deficiency revealed dysregulation of immunity-related genes in both species. To phenotypically validate the transcriptomic results, we challenged zebrafish coilin mutants with lipopolysaccharide (LPS), which triggers an innate immune response, and identified an attenuated response to LPS in vivo in the zebrafish coilin mutants. Our results support a vital novel function for coilin in vertebrates in regulating the expression of immunity-related genes. Moreover, these findings could lead to more research on how coilin regulates innate immunity in animals and humans.

Coilin is a nucleoplasmic protein that is enriched in some cell types in the Cajal body (CB). CBs take part in the biogenesis of many different types of ribonucleoproteins (RNPs), such as small nuclear RNPs. Coilin is known as the CB marker protein and is required for CB formation. The function of nucleoplasmic coilin is less understood and has been shown to impact protein modification by SUMO, the small ubiquitin-like modifier. Additionally, it is known that coilin is recruited to sites of DNA damage caused by UVA exposure or expression of herpes simplex viral protein. PARP1, a DNA damage response protein, has been shown to be SUMOylated by PIAS4, a SUMO E3 ligase that associates with coilin. Here, we show that SUMOylation of PARP1 is lessened when coilin is suppressed. We also found that coilin knockdown and a SUMO inhibitor drug, TAK-981, influence the dynamics of PARP1 in response to micro-irradiation. Additionally, we find that the SUMOylation status of coilin influences its mobility in the CB and recruitment to sites of DNA damage. These data demonstrate that coilin and SUMOylation both have an influence on the DNA damage response.

Cajal bodies (CBs) are membraneless organelles whose mechanism of formation is still not fully understood. Many proteins contribute to the formation of CBs, including Nopp140 (NOLC1), WRAP53 and coilin. Coilin is modified on multiple different lysine residues by SUMO, the small ubiquitin-like modifier. In addition to its accumulation in CBs, coilin is also found in the nucleoplasm, where its role is still being evaluated. Here, we demonstrate a novel mechanism of CB regulation by examining the interaction changes of coilin when its SUMOylation is disrupted. The impact of global SUMOylation inhibition and targeted disruption of coilin SUMOylation on CB formation was examined. We found that two types of global SUMOylation inhibition and expression of SUMO-deficient coilin mutants increased CB number but decreased CB size. Additionally, we saw via coimmunoprecipitation that a SUMO-deficient coilin mutant has altered interaction with Nopp140. This demonstrates increased mechanistic ties between CB formation and SUMOylation.

Cajal bodies (CBs) are subnuclear domains that contribute to the biogenesis of several different classes of ribonucleoproteins (RNPs), including small nuclear RNPs. Only some cell types contain abundant CBs, such as neuronal cells and skeletal muscle, but CBs are invariant features of transformed cells. In contrast, coilin, the CB marker protein, is a ubiquitously expressed nuclear protein, but the function of coilin in cell types that lack CBs is not well understood. We have previously shown that coilin promotes microRNA biogenesis by promoting phosphorylation of DGCR8, a component of the microprocessor. Here, we identify seven additional residues of DGCR8 with decreased phosphorylation upon coilin knockdown. In addition to phosphorylation, the addition of a small ubiquitin-like modifier (SUMO) to DGCR8 also increases its stability. Because of coilin’s role in the promotion of DGCR8 phosphorylation, we investigated whether coilin is involved in DGCR8 SUMOylation. We show that coilin knockdown results in global decrease of protein SUMOylation, including decreased DGCR8 and Sp100 (a PML body client protein) SUMOylation and decreased SMN expression. Alternatively, we found that coilin expression rescued Sp100 SUMOylation and increased DGCR8 and SMN levels in a coilin knockout cell line. Furthermore, we found that coilin facilitates RanGAP1 SUMOylation, interacts directly with components of the SUMOylation machinery (Ubc9 and SUMO2), and, itself, is SUMOylated in vitro and in vivo . In summary, we have identified coilin as a regulator of DGCR8 phosphorylation and a promotor of protein SUMOylation with SUMO E3 ligase-like activity.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression. An important step in miRNA biogenesis occurs when primary miRNAs are bound and cleaved by the microprocessor to generate precursor miRNAs. Regulation at this step is essential and one such regulator includes m6A RNA methylation, an RNA modification found on primary miRNAs that is installed by METTL3 and bound by hnRNPA2B1. Our lab has recently discovered that the Cajal body marker protein coilin also participates in miRNA biogenesis and hypothesized that coilin may be influencing miRNA biogenesis through m6A RNA methylation. Here we report that coilin suppression reduces m6A on primary Let7a and miR-21. We also found that coilin suppression reduced the protein expression of hnRNPA2B1 and METTL3. We observed an interaction between coilin and ectopically expressed METTL3 and found that coilin suppression reduced the nucleoplasmic portion of METTL3 and blunted ectopic METTL3 phosphorylation. Finally, coilin suppression disrupted the greater METTL3 complex with cofactors METTL14 and WTAP. Collectively, our work has uncovered a role for coilin in mediating m6A RNA methylation and provides an avenue by which coilin participates in miRNA biogenesis.

Cajal bodies (CBs) are membraneless subnuclear domains that contribute to the biogenesis and maturation of ribonucleoproteins (RNPs) which take part in fundamental cellular activities such as translation, pre‐mRNA splicing, and telomere maintenance. Partitioning of CBs occurs through liquid‐liquid phase separation (LLPS) where the CB marker protein, coilin, is thought to scaffold the CB. However, the formation of CBs and the regulation of CB number has not been fully elucidated. Disease states such as cancer and preeclampsia have altered CB expression, thus additional insight into CB condensation would provide an important cellular model system that could be used to investigate these and other diseases. We hypothesize that the post‐translational modification (PTM) of coilin regulates CB formation and number by influencing the LLPS of coilin via residues in its intrinsically disordered region (IDR). Here we show, for the first time, a cycloheximide inducible model of CB formation in fibroblasts without increasing coilin concentration. We are also able to induce CB formation by inhibiting kinase GSK3β with indirubin, as well as abolish CB formation by inhibiting phosphatases PP1 and PP2A with okadaic acid. Lastly, we have identified two phase‐separating domains in coilin’s IDR using the phase separation predicting software ParSe. Deletion of each domain prevents CB formation in HeLa cells but does not prevent coilin’s self‐interaction. Collectively, these data will be able to help us understand the role of coilin’s scaffolding capabilities via LLPS mediated CB condensation, as well as provide an important cellular model system that can be used to investigate CB functionality in disease states where CB formation is dysregulated.

Cajal bodies (CBs) are nuclear bodies that are hosts for the modification and assembly of ribonucleoproteins (RNPs). The nuclear protein coilin is known as the CB marker protein as it is the scaffold for CB assembly and vital for structural integrity. However, there is still not much understood about the function of coilin. Recent studies by our lab implicate coilin in the biogenesis of microRNAs (miRNAs). Currently, there are sparse options in animal models for studying coilin and CB function that account for limitations such as off‐target effects of coilin depletion or incomplete protein knockdown. Due to this, we sought to develop novel coilin mutations in zebrafish to advance functional studies of this protein and CBs. In considering how we would approach this, we opted for the innovative CRISPR/Cas9 technology as an optimal strategy and hypothesized that the implementation of this genome editing technology would allow us to develop multiple lines of coilin‐modified zebrafish for studies investigating coilin’s role in miRNA biogenesis. We selected a suitable Protospacer Adjacent Motif (PAM) sequence and designed guide RNAs for our mutations of interest in silico, which we then generated in vitro, and injected into fertilized zebrafish embryos at the one‐cell stage. An initial screening for insertions or deletions (indels) took place at 72 hours post fertilization to determine mutation frequency and once again when the zebrafish reached adulthood. Through our efforts, we have managed to obtain zebrafish that are heterozygous for coilin‐null mutations and are currently in the midst of screening for additional lines of coilin mutations which could serve to characterize the functional domains of coilin in vivo. Utilizing CRISPR/Cas9 technology, we have generated the first coilin‐null mutation in a zebrafish line allowing our lab to expand our investigations into the role of coilin and CBs in the regulatory network of the nucleus.

Coilin was first identified as the marker protein for Cajal Bodies (CBs). However, CBs are only abundant in neuronal, embryonic, and cancer cells, while primary cells have few CBs. Even in cells with CBs, 70% of coilin is nucleoplasmic, not found in CBs. CBs are subnuclear domains that function in small nuclear ribonucleoprotein (snRNP) biogenesis as well as production of telomerase RNP, expression and 3'end processing of small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and small CB-associated RNAs (scaRNAs). The function of nucleoplasmic coilin is not well understood. We have previously shown that coilin promotes miRNA biogenesis by promoting phosphorylation of the Microprocessor component, DGCR8, at its S377 residue in both cells with and without CBs. We have since identified 7 additional serine residues of DGCR8 with decreased phosphorylation in coilin knockdown through phosphoproteomic analysis of HeLa cells. In addition to phosphorylation, the addition of a small ubiquitin-like modifier (SUMO), or SUMOylation, of DGCR8 also increases its stability. Because of coilin's role in the promotion of one post-translational modification, here we aimed to test the hypothesis that coilin knockdown alters protein SUMOylation and results in altered nuclear organization. We conducted a series of siRNA knockdowns, Western blots, and immunoprecipitation experiments. Here we show, for the first time, that coilin knockdown results in global decrease of protein SUMOylation in cells with (HeLa) and without CBs (WI-38), suggesting that regulation of protein SUMOylation may be a function of nucleoplasmic coilin. Specifically, we found that coilin knockdown results in decreased SUMOylation of DGCR8 and Speckled protein 100kDa (Sp100), a major component of the promyelocytic leukemia nuclear body (PML-NB). Sp100 is vital to PML-NB formation, and decreased SUMOylation of Sp100 decreases its stability. This information led us to the second part of our hypothesis. We used immunofluorescence and found that coilin knockdown results in decreased numbers of PML-NBs per cell. To summarize, we have identified coilin as a regulator of protein phosphorylation, SUMOylation, and nuclear organization.

The nuclear factor-Kappa B (NF-κB) pathway is a crucial mediator of inflammatory signaling. Aberrant activation of NF-κB is associated with several disorders including preeclampsia (PE). Many regulators of the NF-κB pathway have been identified, including microRNAs (miRNAs). Specifically, miR-517-3p targets mRNA encoding TNFAIP3 Interacting Protein 1 (TNIP1), an inhibitor of NF-κB signaling. Activation of NF-κB increases production of the cytokine TNF superfamily member 15 (TNFSF15), leading to the upregulation of anti-angiogenic soluble vascular endothelial growth factor receptor 1 (sFlt-1). We have previously observed that Cajal bodies (CBs), subnuclear domains, are associated with the chromosome 19 miRNA gene cluster (C19MC), which encodes miR-517-3p. We have also found that coilin, the CB marker protein, is a positive regulator of miRNA biogenesis. Here we report that coilin is a regulator of miR-517-3p, sFlt-1, TNIP1, TNFSF15 and NF-κB activation, and this regulation is influenced by hypoxia. We also report that coilin and CBs are induced in the reduced uterine perfusion pressure (RUPP) rat model of PE. Collectively, the data presented here implicate coilin as a novel regulator of NF-κB activation and sFlt-1 upregulation.

The monofunctional platinum(II) complex, phenanthriplatin, acts by blocking transcription, but its regulatory effects on long-noncoding RNAs (lncRNAs) have not been elucidated relative to traditional platinum-based chemotherapeutics, e.g., cisplatin. Here, we treated A549 non-small cell lung cancer and IMR90 lung fibroblast cells for 24 h with either cisplatin, phenanthriplatin or a solvent control, and then performed microarray analysis to identify regulated lncRNAs. RNA22 v2 microRNA software was subsequently used to identify microRNAs (miRNAs) that might be suppressed by the most regulated lncRNAs. We found that miR-25-5p, -30a-3p, -138-5p, -149-3p, -185-5p, -378j, -608, -650, -708-5p, -1253, -1254, -4458, and -4516, were predicted to target the cisplatin upregulated lncRNAs, IMMP2L-1, CBR3-1 and ATAD2B-5, and the phenanthriplatin downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1. Then, we used qRT-PCR to measure the expression of miR-25-5p, -378j, -4516 (A549) and miR-149-3p, -608, and -4458 (IMR90) to identify distinct signaling effects associated with cisplatin and phenanthriplatin. The signaling pathways associated with these miRNAs suggests that phenanthriplatin may modulate Wnt/β-catenin and TGF-β signaling through the MAPK/ERK and PTEN/AKT pathways differently than cisplatin. Further, as some of these miRNAs may be subject to dissimilar lncRNA targeting in A549 and IMR90 cells, the monofunctional complex may not cause toxicity in normal lung compared to cancer cells by acting through distinct lncRNA and miRNA networks.

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is composed of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of coimmunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.

Hypoxia is a severe stressor to cellular homeostasis. At the cellular level, low oxygen triggers the transcription of a variety of genes supporting cell survival and oxygen homeostasis mediated by transcription factors, such as hypoxia-inducible factors (HIFs). Among many determinants dictating cell responses to hypoxia and HIFs are microRNAs (miRNAs). Cajal bodies (CBs), subnuclear structures involved in ribonucleoprotein biogenesis, have been recently proven to contribute to miRNA processing and biogenesis but have not been studied under hypoxia. Here, we show, for the first time, a hypoxia-dependent increase in CB number in WI-38 primary fibroblasts, which normally have very few CBs. Additionally, the CB marker protein coilin is upregulated in hypoxic WI-38 cells. However, the hypoxic coilin upregulation was not seen in transformed cell lines. Furthermore, we found that coilin is needed for the hypoxic induction of a well-known hypoxia-induced miRNA (hypoxamiR), miR-210, as well as for the hypoxia-induced alternative splicing of the miR-210 host gene, MIR210HG. These findings provide a new link in the physiological understanding of coilin, CBs and miRNA dysregulation in hypoxic pathology.

Cajal bodies (CBs) are subnuclear domains involved in the formation of ribonucleoproteins (RNPs) including small nuclear RNPs (snRNPs). CBs associate with specific gene loci, which impacts expression and provides a platform for the biogenesis of the nascent transcripts emanating from these genes. Here we report that CBs can associate with the C19MC microRNA (miRNA) gene cluster, which suggests a role for CBs in the biogenesis of animal miRNAs. The machinery involved in the formation of miRNAs includes the Drosha/DGCR8 complex, which processes primary-miRNA to precursor miRNA. Further processing of precursor miRNA by Dicer and other components generates mature miRNA. To test if CBs influence the expression and formation of miRNAs, we examined two representative miRNAs (miR-520 h and let-7a) in conditions that disrupt CBs. CB disruption correlates with alterations in the level of primary and mature miRNA and the let-7a mRNA target, HMGA2. We have also found that the processing of some small CB-specific RNAs (scaRNAs) is directly mediated by the Drosha/DGCR8 complex. ScaRNAs form scaRNPs, which play an important role in snRNP formation. Collectively, our results demonstrate that CBs and the miRNA processing machinery functionally interact and together contribute to the biogenesis of miRNAs and snRNPs.

The Cajal body (CB) is a subnuclear domain that participates in the biogenesis of many different types of ribonucleoproteins (RNPs), including small nuclear RNPs (snRNPs), small Cajal body-specific RNPs (scaRNPs) and telomerase. Most scaRNAs, the RNA component of scaRNPs, accumulate in CBs. However, there are three scaRNAs (scaRNA 2, 9, and 17) that are known to be processed into small, nucleolar-enriched fragments. Evidence suggests that these fragments are packaged into a new class of RNPs, called regulatory RNPs (regRNPs), and may modify small nucleolar RNP (snoRNP) activity, thus playing a role in rRNA modification. However, the mechanism by which these fragments are produced is unknown. Previous work has reported the involvement of Drosha and DGCR8 in the cleavage of primary-scaRNA9. Here, we expand on that knowledge by identifying sequence elements necessary for the efficient production of these RNA fragments and demonstrate that primary scaRNA 2 and 17 are also processed by the Drosha-DGCR8 complex. Collectively, our work establishes new factors in the scaRNP biogenesis pathway and adds to the ever-expanding list of noncanonical functions for the microprocessor complex.

The most common types of modification in human rRNA are pseudouridylation and 2'-O ribose methylation. These modifications are performed by small nucleolar ribonucleoproteins (snoRNPs) which contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. rRNA modifications can vary, generating ribosome heterogeneity. One possible method that can be used to regulate rRNA modifications is by controlling snoRNP activity. RNA fragments derived from some small Cajal body-specific RNAs (scaRNA 2, 9 and 17) may influence snoRNP activity. Most scaRNAs accumulate in the Cajal body - a subnuclear domain - where they participate in the biogenesis of small nuclear RNPs, but scaRNA 2, 9 and 17 generate nucleolus-enriched fragments of unclear function, and we hypothesize that these fragments form regulatory RNPs that impact snoRNP activity and modulate rRNA modifications. Our previous work has shown that SMN, Drosha and various stresses, including etoposide treatment, may alter regulatory RNP formation. Here we demonstrate that etoposide treatment decreases the phosphorylation of SMN, reduces Drosha levels and increases the 2'-O-methylation of two sites within 28S rRNA. These findings further support a role for SMN and Drosha in regulating rRNA modification, possibly by affecting snoRNP or regulatory RNP activity.

Ribosomes can be heterogeneous, and the major contributor to ribosome heterogeneity is variation in rRNA modification. There are two major types of rRNA modification, pseudouridylation and ribose methylation. In humans, the majority of these rRNA modifications are conducted by two classes of small nucleolar ribonucleoproteins (snoRNPs), which contain a guide RNA (small nucleolar RNA, snoRNA) complexed with proteins. Box H/ACA snoRNPs conduct pseudouridylation modifications and box C/D snoRNPs generate ribose methylation modifications. It is unclear how ribosome heterogeneity is accomplished in regards to the understanding of the signals and factors that regulate rRNA modifications. We have recently reported that a new class of RNP, that we term regulatory RNP (regRNP), may contribute to rRNA modification as well as the modification of nucleolar trafficked U6 snRNA, via interactions with snoRNPs. Here we report the identification of additional regRNP activities that influence the methylation of two sites within 18S rRNA, two sites within 28S rRNA and one site within U6 snRNA. These findings provide additional proof that regulation of snoRNP activity contributes to ribosome heterogeneity.

Small Cajal body-specific RNAs (scaRNAs) are part of small Cajal body-specific ribonucleoproteins (scaRNPs) that modify small nuclear RNA (snRNA) in Cajal bodies (CBs). Several scaRNAs (scaRNA 2, 9 and 17) have been found to generate smaller, nucleolus-enriched fragments. We hypothesize that the fragments derived from scaRNA 2, 9 and 17 form regulatory RNPs that influence the level of modifications within rRNA by altering small nucleolar RNP (snoRNP) activity. Here we show that external factors such as DNA damaging agents can alter the scaRNA9 full length to processed fragment ratio. We also show that full-length scaRNA2 levels are likewise impacted by DNA damage, which correlates with the disruption of SMN, coilin and WRAP53 co-localization in CBs. The dynamics of scaRNA9 were also shown to be affected by Drosha levels, which suggests that this protein may participate in the biogenesis and processing of this non-coding RNA. Identification of factors that contribute to scaRNA 2, 9 and 17 processing may facilitate an assessment of how external stress can lead to changes in rRNA modifications.

DATA ACCESS The cDNA sequences reported in this paper have been deposited in the GenBank database (accession numbers): KF175165 - KF175225 and BACs accession numbers that are used in this study: AC148621 , AC190226 , AC097327 , AC097332 , AC190226 , AC097333 , AC145401 , AC187943 , AC166855 , AC166597 , AC167295 , AC235773 , AC202644 , and AC234805 . ABSTRACT The burst of segmental duplications during human and great ape evolution focuses on a set of “core” duplicons encoding great-ape-specific gene families. Characterization of these gene families is complicated by their high copy number, incomplete sequence, and polymorphic nature. We investigate the structure, transcriptional diversity, and protein localization of the nuclear pore complex-interacting protein (NPIP) or Morpheus gene family. The corresponding core, LCRA, encodes one of the most rapidly evolving genes in the human genome; LCRA has expanded to ~20 copies from a single ancestral locus in Old World monkey and is associated with most of the recurrent chromosome 16 microdeletions implicated in autism and mental retardation. Phylogenetic analysis and cDNA sequencing suggest two distinct subfamilies or subtypes, NPIPA and NPIPB. The latter expanded recently within the great apes due to a series of structural changes within the canonical gene structure. Among Old World monkey, we observe a testis-specific pattern of expression that contrasts with the ubiquitous pattern observed among human tissues. This change in the expression profile coincides with the structural events that reshaped the structure and organization of the gene family. Most of the expressed human copies are capable of producing an open reading frame. Immunofluorescence analyses of the morpheus genes showed a primary localization to both the nucleus and its periphery. We show that morpheus genes may be upregulated upon pI:C treatment and find evidence of human autoantibodies produced against the NPIPB protein, raising the possibility that morpheus genes may be related to immune- or autoimmune-related function.

The biogenesis of small nuclear ribonucleoproteins (snRNPs), small Cajal body-specific RNPs (scaRNPs), small nucleolar RNPs (snoRNPs) and the telomerase RNP involves Cajal bodies (CBs). Although many components enriched in the CB contain post-translational modifications (PTMs), little is known about how these modifications impact individual protein function within the CB and, in concert with other modified factors, collectively regulate CB activity. Since all components of the CB also reside in other cellular locations, it is also important that we understand how PTMs affect the subcellular localization of CB components. In this review, we explore the current knowledge of PTMs on the activity of proteins known to enrich in CBs in an effort to highlight current progress as well as illuminate paths for future investigation.