Ongoing U snRNP Biogenesis Is Required for the Integrity of Cajal Bodies

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.

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Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

Neurology International ◽

10.3390/neurolint14010002 ◽

2021 ◽

Vol 14 (1) ◽

pp. 11-33

Author(s):

Sui Sawaguchi ◽

Kenji Tago ◽

Hiroaki Oizumi ◽

Katsuya Ohbuchi ◽

Masahiro Yamamoto ◽

...

Keyword(s):

Protein Expression ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Mtor Signaling ◽

Marker Protein ◽

Wild Type ◽

Mutant Proteins ◽

Oligodendroglial Cell ◽

Hypomyelinating Leukodystrophy ◽

In Cells

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

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RNA-mediated interaction of Cajal bodies and U2 snRNA genes

The Journal of Cell Biology ◽

10.1083/jcb.200105084 ◽

2001 ◽

Vol 154 (3) ◽

pp. 499-510 ◽

Cited By ~ 73

Author(s):

Mark R. Frey ◽

A. Gregory Matera

Keyword(s):

Nuclear Export ◽

Marked Decrease ◽

Gene Clusters ◽

Cajal Bodies ◽

U2 Snrna ◽

Intragenic Deletions ◽

High Concentrations ◽

Nuclear Structures ◽

Insight Into ◽

Small Nuclear Ribonucleoproteins

Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

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Subcellular localization of protein kinase C δ and ε affects transcriptional and post-transcriptional processes in four-cell mouse embryos

Reproduction ◽

10.1530/rep.1.00572 ◽

2005 ◽

Vol 130 (4) ◽

pp. 453-465 ◽

Cited By ~ 8

Author(s):

Hesam Dehghani ◽

Cara Reith ◽

Ann C Hahnel

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Subcellular Localization ◽

Cajal Bodies ◽

Message Processing ◽

Mrna Transcription ◽

Cell Stage ◽

Kinase C ◽

Sm Protein ◽

Small Nuclear Ribonucleoproteins

During mouse preimplantation development, two isozymes of protein kinase C (PKC), δ and ε, transiently localize to nuclei at the early four-cell stage. In order to study their functions at this stage, we altered the subcellular localization of these isozymes (ratio of nuclear to cytoplasmic concentrations) with peptides that specifically activate or inhibit translocation of each isozyme. The effects of altering nuclear concentration of each isozyme on transcription (5-bromouridine 5′-triphosphate (BrUTP) incorporation), amount and distribution of small nuclear ribonucleoproteins (snRNPs), nucleolar dynamics (immunocytochemistry for Smith antigen (Sm) protein) and the activity of embryonic alkaline phosphatase (EAP; histochemistry) were examined. We found that nuclear concentration of PKC ε correlated with total mRNA transcription. Higher nuclear concentrations of both PKC δ and ε decreased storage of snRNPs in Cajal bodies and decreased the number of nucleoli, but did not affect the nucleoplasmic concentration of snRNPs. Inhibiting translocation of PKC δ out of the nucleus at the early four-cell stage decreased cytoplasmic EAP activity, whereas inhibiting translocation of PKC ε increased EAP activity slightly. These results indicate that translocation of PKC δ and ε in and out of nuclei at the early four-cell stage in mice can affect transcription or message processing, and that sequestration of these PKC in nuclei can also affect the activity of a cytoplasmic protein (EAP).

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PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

10.1101/160739 ◽

2017 ◽

Author(s):

Beata Jonik-Nowak ◽

Thomas Menneteau ◽

Didier Fesquet ◽

Véronique Baldin ◽

Catherine Bonne-Andrea ◽

...

Keyword(s):

Cell Proliferation ◽

Cellular Stress Response ◽

Cajal Body ◽

Cajal Bodies ◽

Central Component ◽

20S Proteasome ◽

Independent Manner ◽

Nuclear Dynamics ◽

Multiple Functions ◽

In Cells

ABSTRACTPA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However its exact mechanisms of action are unclear and likely to involve additional partners that remain to be identified. Here we report the identification of the first cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal Bodies by inhibition of its association with the key Cajal body component coilin. Altogether, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.Significance StatementThe 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which a nuclear complex called PA28γ. In particular, PA28γ participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a novel protein, PIP30/FAM192A, which binds tightly to PA28γ and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus PIP30/FAM192A critically controls the interactome and consequently the functions of PA28γ, and appears to be a new player in the fine regulation of intracellular proteostasis in the cell nucleus.

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Synergistic interactions between Cajal bodies and the miRNA processing machinery

Molecular Biology of the Cell ◽

10.1091/mbc.e20-02-0144 ◽

2020 ◽

Vol 31 (15) ◽

pp. 1561-1569

Author(s):

Madelyn K. Logan ◽

Douglas M. McLaurin ◽

Michael D. Hebert

Keyword(s):

Cajal Bodies ◽

Mirna Processing ◽

Synergistic Interactions ◽

Processing Machinery ◽

Small Nuclear Ribonucleoproteins

Our results demonstrate that Cajal bodies and the microRNA (miRNA) processing machinery functionally interact and together contribute to the biogenesis of miRNAs and small nuclear ribonucleoproteins.

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siRNA-Mediated Silencing of αII Spectrin Gene Expression in Cells Containing DNA Interstrand Cross-Links and Its Relationship to Fanconi Anemia.

Blood ◽

10.1182/blood.v104.11.725.725 ◽

2004 ◽

Vol 104 (11) ◽

pp. 725-725

Author(s):

Muriel W. Lambert ◽

Laura W. McMahon ◽

Joel A. Lefferts ◽

Deepa M. Sridharan

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Repair Process ◽

Cross Linking ◽

Transfected Cells ◽

Interstrand Cross Links ◽

Cross Links ◽

Nuclear Foci ◽

In Cells

Abstract Fanconi anemia (FA) is characterized by bone marrow failure, a predisposition to cancer and, at the cellular level, a hypersensitivity to DNA interstrand cross-linking agents that correlates with a defect in ability to repair cross-links produced by these agents. We have previously shown that the structural protein, nonerythroid a spectrin (αSpII∑*), binds to DNA containing interstrand cross-links and plays an important role in the repair of this type of damage and that there is a deficiency in this protein in cells from FA-A, FA-B, FA-C, FA-D1, FA-F and FA-G cells. We have also shown that after damage to normal cells, αSpII∑* co-localizes in nuclear foci with FANCA, FANCF and the DNA interstrand cross-link repair protein XPF. In FA-A cells, where there is a deficiency in αSpII∑*, damage-induced nuclear foci formation is significantly reduced. In order to further assess the functional importance of αSpII∑* in the repair process and the DNA repair defect in FA cells, studies were carried out on siRNA-mediated silencing of αII spectrin gene expression. Three siRNA oligonucleotide templates were synthesized, which were targeted for different αII spectrin gene sequences. The 21-mer siRNAs produced were purified and normal human lymphoblastoid cells were transfected with these siRNAs. αII spectrin mRNA and protein levels in transfected cells were determined by RT-PCR and Western blot analysis, respectively. By 48 hours after transfection, levels of αII spectrin mRNA and protein were approximately 34% and 30% of mock transfected cells, respectively. Cells were then damaged with 8-MOP plus UVA light so as to produce DNA interstrand cross-links and localization of αII spectrin and FANCA in the nuclei was examined by immunofluorescence microscopy. The results showed that, in cells transfected with αII spectrin siRNA, there was a marked reduction in the number of αII spectrin and FANCA foci in the nuclei of the siRNA transfected cells compared to the mock transfected cells. There was also markedly reduced survival of the siRNA transfected cells after damage compared to mock transfected cells. These results show that there is a correlation between a reduction of αII spectrin levels in these cells and decreased formation of FANCA as well as αII spectrin nuclear foci after damage with a DNA interstrand cross-linking agent and that this in turn correlates with decreased survival and DNA repair in these cells after DNA damage. These results support our model that αII spectrin is needed in the DNA repair process where it acts as a scaffold in the recruitment and alignment of FANC and repair proteins at sites of DNA damage and that, in FA cells, where there is a deficiency in αII spectrin, this recruitment and repair are defective. α∑

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Rice stripe virus p2 Colocalizes and Interacts with Arabidopsis Cajal Bodies and Its Domains in Plant Cells

BioMed Research International ◽

10.1155/2020/5182164 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Luping Zheng ◽

Pengxiang Hong ◽

Xiaonan Guo ◽

Yang Li ◽

Li Xie

Keyword(s):

Plant Virus ◽

Plant Cells ◽

Rice Stripe Virus ◽

Bimolecular Fluorescence Complementation ◽

Cajal Bodies ◽

Marker Protein ◽

Yeast Two Hybrid ◽

Systemic Movement ◽

And Function ◽

Two Hybrid

p2 of rice stripe virus may translocate from the nucleus to the cytoplasm and recruit nucleolar functions to promote virus systemic movement. Cajal bodies (CBs) are nuclear components associated with the nucleolus, which play a major role in plant virus infection. Coilin, a marker protein of CBs, is essential for CB formation and function. Coilin contains three domains, the N-terminal, the center, and the C-terminal fragments. Using yeast two-hybrid, colocalization, and bimolecular fluorescence complementation (BiFC) approaches, we show that p2 interacts with the full-length of Arabidopsis thaliana coilin (Atcoilin), the center and C-terminal domain of Atcoilin in the nucleus. Moreover, the N-terminal is indispensable for Atcoilin to interact with Cajal bodies.

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Impaired DNA Damage Response in Cells Expressing an Exon 11-Deleted Murine Brca1 Variant That Localizes to Nuclear Foci

Molecular and Cellular Biology ◽

10.1128/mcb.21.12.4005-4015.2001 ◽

2001 ◽

Vol 21 (12) ◽

pp. 4005-4015 ◽

Cited By ~ 69

Author(s):

L. Julie Huber ◽

Thomas W. Yang ◽

Christopher J. Sarkisian ◽

Stephen R. Master ◽

Chu-Xia Deng ◽

...

Keyword(s):

Dna Damage ◽

Nuclear Localization ◽

Full Length ◽

Null Alleles ◽

Brca1 Protein ◽

Nuclear Localization Signals ◽

Nuclear Foci ◽

Exon 11 ◽

Homozygous Deletions ◽

In Cells

ABSTRACT Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Δ11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Δ11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Δ11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Δ11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Δ11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Δ11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Δ11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.

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Small Cajal Body–specific RNAs of Drosophila Function in the Absence of Cajal Bodies

Molecular Biology of the Cell ◽

10.1091/mbc.e09-09-0777 ◽

2009 ◽

Vol 20 (24) ◽

pp. 5250-5259 ◽

Cited By ~ 48

Author(s):

Svetlana Deryusheva ◽

Joseph G. Gall

Keyword(s):

Fluorescent In Situ Hybridization ◽

Xenopus Oocyte ◽

Cajal Body ◽

Cajal Bodies ◽

Marker Protein ◽

Wild Type ◽

Guide Rnas ◽

Small Nuclear Rnas ◽

Covalent Modifications

During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.

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Two Splice Variants of Nopp140 in Drosophila melanogaster

Molecular Biology of the Cell ◽

10.1091/mbc.01-04-0162 ◽

2002 ◽

Vol 13 (1) ◽

pp. 362-381 ◽

Cited By ~ 19

Author(s):

John M. Waggener ◽

Patrick J. DiMario

Keyword(s):

Hela Cell ◽

Rna Binding ◽

Rna Binding Proteins ◽

Splice Variants ◽

Cajal Bodies ◽

Carboxy Terminus ◽

Cell Nuclei ◽

Rich Domain ◽

Similar Phase ◽

In Cells

The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophilavariants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, bothDrosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies inXenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140ΔRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.

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