Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SC-35 is a non-snRNP spliceosome component that is specifically recognized by the anti-spliceosome monoclonal antibody alpha SC-35. In this paper we provide direct evidence that SC-35 is an essential splicing factor and we examine the immunolocalization of SC-35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC-35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC-35 speckled staining pattern is lost during prophase and speckles containing SC-35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC-35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.
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PMID:Associations between distinct pre-mRNA splicing components and the cell nucleus. 183 87

Protein 4.1, originally identified as a component of the membrane-skeleton of the red blood cell, has also been localized in the nucleus of mammalian cells. To learn more about nuclear 4.1 protein, we have analyzed the nature of its association with the nuclear structure in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells were digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens were extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembled that of SC35. Experiments carried out in transcriptionally active nuclei showed that protein 4.1 distributed in irregularly shaped speckles which appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulated in rounded speckles lacking interconnections. When cells were released from transcriptional inhibition, protein 4.1 redistributed back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Finally, coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens.
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PMID:Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains. 904 54

Protein tyrosine phosphatases are involved in the regulation of important cellular processes such as signal transduction, cell cycle progression, and tumor suppression. Here we report the cloning and characterization of PIR1, a novel member in the dual-specificity phosphatase subfamily of the protein tyrosine phosphatases. PIR1 also contains two stretches of arginine-rich sequences. We have shown that the recombinant PIR1 protein possessed an intrinsic phosphatase activity on phosphotyrosine-containing substrate. A unique feature of this phosphatase is that it binds directly to RNA in vitro with high affinity. In addition, we have found that PIR1 interacted with splicing factors 9G8 and SRp30C, possibly through an RNA intermediate during a yeast two-hybrid screen. PIR1 exhibited a nuclear-staining pattern that was sensitive to RNase A, but not to DNase I, suggesting that PIR1 in the cells are associated with RNA and/or ribonucleoprotein particles. Furthermore, a fraction of PIR1 showed a speckle-staining pattern that superimposed with that of the splicing factor, SC35. Taken together, our data suggest that PIR1 is a novel phosphatase that may participate in nuclear mRNA metabolism.
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PMID:PIR1, a novel phosphatase that exhibits high affinity to RNA . ribonucleoprotein complexes. 968 86

A protein (SNP70) has been isolated that binds to the Src homology domain 3 of p47(phox), p85alpha, and c-src. Cloning and sequencing of the polypeptide revealed it to be a 70-kDa protein that has a number of potential domains, including Src homology 3 binding motifs and several nuclear localization signals. Immunofluorescence using anti-peptide antibodies revealed SNP70 to be primarily concentrated in the nucleus but excluded from nucleoli, in interphase cells. However, it was distributed throughout the cytoplasm in dividing cells. Extraction and subfractionation experiments indicated that SNP70 did not bind directly to DNA but did bind to poly(G)-rich oligonucleotides and was resistant to extraction with non-ionic detergents but was solubilized by treatment with RNase, high salt, or ammonium sulfate. Double-immunofluorescence experiments showed that SNP70 co-localized with two pre-mRNA splicing factors SC35 and U2B" within the nucleus. A population of SNP70 was found outside the nucleus, and double-immunofluorescence and immunoelectron microscopy demonstrated that it associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. The data suggest that SNP70 associates with nuclear or perinuclear filaments and may play a role in the regulation of pre-mRNA processing.
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PMID:A nuclear SH3 domain-binding protein that colocalizes with mRNA splicing factors and intermediate filament-containing perinuclear networks. 1137 89

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre-mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins-1 and -3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin-specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two-dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin-1, whereas galectins-3, -5, and -7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin-1. Colocalization of the galectin-1-dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin-1-binding sites expressed DeltaNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin-1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki-67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.
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PMID:New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites. 1463 Mar 91

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.
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PMID:Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles. 1551 Dec 25

The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.
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PMID:Dynamic expression of the RNA-binding protein Sam68 during mouse pre-implantation development. 1832 92

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.
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PMID:SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. 1939 95

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.
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PMID:C3G dynamically associates with nuclear speckles and regulates mRNA splicing. 2949 66