FLASH is an essential component of Cajal bodies.
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ABSTRACT:
SUBMITTER: Barcaroli D
PROVIDER: S-EPMC1578500 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cajal bodies are specialized and dynamic compartments in the nucleus that are involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Because of the dynamic and varied roles of Cajal bodies, it is of great interest to identify the components of Cajal bodies to better understand their functions. We performed a genome-wide screen to identify proteins that colocalize with coilin, the marker protein of Cajal bodies. In this study, we identified and characterized Fam118B as a newly discovered component of Cajal bodies. Fam118B is widely expressed in a variety of cell lines derived from various origins. Overexpression of Fam118B changes the canonical morphology of Cajal bodies, whereas depletion of Fam118B disrupts the localization of components of Cajal bodies, including coilin, the survival of motor neuron protein (SMN) and the Sm protein D1 (SmD1, also known as SNRPD1). Moreover, depletion of Fam118B reduces splicing capacity and inhibits cell proliferation. In addition, Fam118B associates with coilin and SMN proteins. Fam118B depletion reduces symmetric dimethylarginine modification of SmD1, which in turn diminishes the binding of SMN to this Sm protein. Taken together, these data indicate that Fam118B, by regulating SmD1 symmetric dimethylarginine modification, plays an important role in Cajal body formation, snRNP biogenesis and cell viability.
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Project description:The WRAP53 gene gives rise to a p53 antisense transcript that regulates p53. This gene also encodes a protein that directs small Cajal body-specific RNAs to Cajal bodies. Cajal bodies are nuclear organelles involved in diverse functions such as processing ribonucleoproteins important for splicing. Here we identify the WRAP53 protein as an essential factor for Cajal body maintenance and for directing the survival of motor neuron (SMN) complex to Cajal bodies. By RNA interference and immunofluorescence we show that Cajal bodies collapse without WRAP53 and that new Cajal bodies cannot be formed. By immunoprecipitation we find that WRAP53 associates with the Cajal body marker coilin, the splicing regulatory protein SMN, and the nuclear import receptor importinβ, and that WRAP53 is essential for complex formation between SMN-coilin and SMN-importinβ. Furthermore, depletion of WRAP53 leads to accumulation of SMN in the cytoplasm and prevents the SMN complex from reaching Cajal bodies. Thus, WRAP53 mediates the interaction between SMN and associated proteins, which is important for nuclear targeting of SMN and the subsequent localization of the SMN complex to Cajal bodies. Moreover, we detect reduced WRAP53-SMN binding in patients with spinal muscular atrophy, which is the leading genetic cause of infant mortality worldwide, caused by mutations in SMN1. This suggests that loss of WRAP53-mediated SMN trafficking contributes to spinal muscular atrophy.
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Project description:[This corrects the article DOI: 10.1371/journal.pbio.1000521.].
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Project description:The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28gamma (proteasome activator subunit gamma). The presence of PA28gamma in coilin-containing complexes is increased by UV-C. Overexpression of PA28gamma, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference-mediated knockdown of PA28gamma attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28gamma as a novel regulator of CB integrity.
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Project description:The mechanisms underlying nuclear body (NB) formation and their contribution to genome function are unknown. Here we examined the non-random positioning of Cajal bodies (CBs), major NBs involved in spliceosomal snRNP assembly and their role in genome organization. CBs are predominantly located at the periphery of chromosome territories at a multi-chromosome interface. Genome-wide chromosome conformation capture analysis (4C-seq) using CB-interacting loci revealed that CB-associated regions are enriched with highly expressed histone genes and U small nuclear or nucleolar RNA (sn/snoRNA) loci that form intra- and inter-chromosomal clusters. In particular, we observed a number of CB-dependent gene-positioning events on chromosome 1. RNAi-mediated disassembly of CBs disrupts the CB-targeting gene clusters and suppresses the expression of U sn/snoRNA and histone genes. This loss of spliceosomal snRNP production results in increased splicing noise, even in CB-distal regions. Therefore, we conclude that CBs contribute to genome organization with global effects on gene expression and RNA splicing fidelity.
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Project description: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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Project description:The ability of most cancer cells to grow indefinitely relies on the enzyme telomerase and its recruitment to telomeres. In human cells, recruitment depends on the Cajal body RNA chaperone TCAB1 binding to the RNA subunit of telomerase (hTR) and is also thought to rely on an N-terminal domain of the catalytic subunit, hTERT. We demonstrate that coilin, an essential structural component of Cajal bodies, is required for endogenous telomerase recruitment to telomeres but that overexpression of telomerase can compensate for Cajal body absence. In contrast, recruitment of telomerase was sensitive to levels of TCAB1, and this was not rescued by overexpression of telomerase. Thus, although Cajal bodies are important for recruitment, TCAB1 has an additional role in this process that is independent of these structures. TCAB1 itself localizes to telomeres in a telomerase-dependent but Cajal body-independent manner. We identify a point mutation in hTERT that largely abolishes recruitment yet does not affect association of telomerase with TCAB1, suggesting that this region mediates recruitment by an independent mechanism. Our results demonstrate that telomerase has multiple independent requirements for recruitment to telomeres and that the function of TCAB1 is to directly transport telomerase to telomeres.
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Project description:We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.
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Project description:Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1-/- embryos and the amount of cytoplasmic snRNP detected in Zpr1-/- embryos was reduced compared with that in wild-type embryos. We found that Zpr1-/- mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2016-03-21 | GSE77917 | GEO