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Biomedical subjects

X D Fu

Publications and source records attributed to X D Fu.

At least 37 records · Page 2Linked to original sources
PubMed1996

[Study on relation of kidney-deficiency with hearing damage and serum trace elements in patients with chronic nephropathy].

The hearing and serum trace element of 73 cases of chronic nephropathy were measured. Results showed that the incidence of hearing damage in high frequency was 68.5% with a mean hypoacusis of 35.1 +/- 6.1 dB, while in 55 patients of Kidney Deficiency type, the incidence of hearing damage in high frequency was 83.6%, it was significantly higher than that in patients without Kidney Deficiency. The serum levels of trace elements (iron and zinc) in patients with Kidney Deficiency were lower than that of normal level and in patients without Kidney Deficiency, and the decrease of serum iron was directly proportional to the degree of hearing damage. There were close relationship between the Kidney Deficiency with the hearing damage as well as the serum levels of iron and zinc.

Adult↗
PubMed1995

Chromosomal localization of mouse and human genes encoding the splicing factors ASF/SF2 (SFRS1) and SC-35 (SFRS2).

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

Alternative Splicing↗
PubMed1995

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation.

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly modify in the three groups, novel structural components were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m3G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hi-bernating dormice, were present in much higher amounts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.

Adrenal Cortex↗
PubMed1995

Genetic selection for balanced retroviral splicing: novel regulation involving the second step can be mediated by transitions in the polypyrimidine tract.

Incomplete splicing is essential for retroviral replication; and in simple retroviruses, splicing regulation appears to occur entirely in cis. Our previous studies, using avian sarcoma virus, indicated that weak splicing signals allow transcripts to escape the splicing pathway. We also isolated a series of avian sarcoma virus mutants in which env mRNA splicing was regulated by mechanisms distinct from those of the wild-type virus. In vitro splicing experiments with one such mutant (insertion suppressor 1 [IS1]) revealed that exon 1 and lariat-exon 2 intermediates were produced (step 1) but the exons were not efficiently ligated (step 2). In this work, we have studied the mechanism of this second-step block as well as its biological relevance. Our results show that the second-step block can be overcome by extending the polypyrimidine tract, and this causes an oversplicing defect in vivo. The requirement for regulated splicing was exploited to isolate new suppressor mutations that restored viral growth by down-regulating splicing. One suppressor consisted of a single U-to-C transition in the polypyrimidine tract; a second included this same change as well as an additional U-to-C transition within a uridine stretch in the polypyrimidine tract. These suppressor mutations affected primarily the second step of splicing in vitro. These results support a specific role for the polypyrimidine tract in the second step of splicing and confirm that, in a biological system, uridines and cytosines are not functionally equivalent within the polypyrimidine tract. Unlike the wild-type virus, the second-step mutants displayed significant levels of lariat-exon 2 in vivo, suggesting a role for splicing intermediates in regulation. Our results indicate that splicing regulation can involve wither the first or second step.

Animals↗
PubMed1994

Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors.

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

Amino Acid Sequence↗
PubMed1994

A serine kinase regulates intracellular localization of splicing factors in the cell cycle.

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells. It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts. Splicing factors redistribute in response to transcription inhibition or viral infection, and nuclear speckles break down and reform as cells progress through mitosis. We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 may have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Amino Acid Sequence↗
PubMed1993

Specific commitment of different pre-mRNAs to splicing by single SR proteins.

Higher eukaryotic cells express a family of essential splicing factors with a characteristic RNA-binding domain and serine/arginine-rich (SR) motif. These SR proteins, which include SC35 and SF2/ASF, are conserved from Drosophila to man, are required for early steps of spliceosome assembly and can influence splice-site selections. To address their mechanisms of action, SR proteins were examined for their role in committing pre-messenger RNA to the splicing pathway. I report here that SC35 was sufficient on its own to form a committed complex with human beta-globin pre-mRNA. Examination of other SR proteins and pre-mRNA substrates revealed that single SR proteins committed different pre-mRNAs to splicing with pronounced substrate specificity. These results suggest that splicing of different pre-mRNAs may require distinct sets of SR proteins, and that the commitment by SR proteins may be a critical step at which alternative and tissue-specific splicing is regulated.

Animals↗
PubMed1993

Novel nuclear autoantigen with splicing factor motifs identified with antibody from hepatocellular carcinoma.

A patient with liver cirrhosis who progressed to hepatocellular carcinoma was found to develop novel antinuclear antibodies. The serum was used to isolate full-length cDNA clones encoding related proteins of 530 amino acids (representative clone HCC1.4) and 524 amino acids (representative clone HCC1.3). Affinity-purified antibodies eluted from recombinant proteins recognized a 64-kD nuclear protein in Western blotting and decorated the nucleoplasm in a speckled-network fashion in immunofluorescence, colocalizing with antibodies to pre-mRNA splicing factor SC35 and uridine-rich small nuclear RNAs. The deduced amino acid sequence contained an arginine/serine-rich (RS) domain and three-ribonucleoprotein consensus sequence domains, two classes of motifs present in several splicing factors. A repeating octapeptide of Arg-Ser-Arg-Ser-Arg(Lys)-Glu(Asp)-Arg-Lys(Arg) was present in RS region of HCC1. This octapeptide sequence called RS-ERK motif was also found in splicing factors U2AF 35- and 65-kD proteins and 70-kD U1 small nuclear ribonucleoprotein. The molecular features and immunolocalization data suggest that the HCC1 autoantigen may be associated with splicing activities and are consistent with observations that autoantibody responses frequently target molecules involved in important cellular biosynthetic functions.

Amino Acid Sequence↗
PubMed1992

General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro.

Animals↗
PubMed1992

Isolation of a complementary DNA that encodes the mammalian splicing factor SC35.

The mammalian splicing factor SC35 is required for the first step in the splicing reaction and for spliceosome assembly. The cloning and characterization of a complementary DNA encoding this protein revealed that it is a member of a family of splicing factors that includes mammalian SF2/ASF. This family of proteins is characterized by the presence of a ribonucleoprotein (RNP)-type RNA binding motif and a carboxyl-terminal serine-arginine-rich (SR) domain. A search of the DNA sequence database revealed that the thymus-specific exon (ET) of the c-myb proto-oncogene is encoded on the antisense strand of the SC35 gene.

Amino Acid Sequence↗
PubMed1992

The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site.

The splicing factor SC35 is required for the first step of the splicing reaction and for the assembly of the earliest ATP-dependent complex detected by native gel electrophoresis (A complex). Here we investigate the role of SC35 in mediating specific interactions between U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) and the 5' and 3' splice sites of pre-mRNA. We show that U1 snRNP interacts specifically with both the 5' and 3' splice sites in the presence of ATP and that SC35 is required for these ATP-dependent interactions. Significantly, the SC35-dependent interaction between U1 snRNP and the 3' splice site requires U2 snRNP but not the 5' splice site. We also show that SC35 is required for the ATP-dependent interaction between U2 snRNP and the branch-point sequence. We conclude that SC35 may play an important role in mediating specific interactions between splicing components bound to the 5' and 3' splice sites.

Adenosine Triphosphate↗
PubMed1991

Associations between distinct pre-mRNA splicing components and the cell nucleus.

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.

Antigens↗
PubMed1991

The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

We previously described an avian sarcoma-leukosis virus (ASLV) insertion mutation that causes a decrease in the ratio of unspliced to spliced RNA in vivo, resulting in a replication defect. Pseudorevertant viruses containing cis-acting suppressor mutations that restored the normal ratio were isolated. One class of the suppressor mutations consists of single-base changes or small deletions near the 3' splice site, while another consists of deletions in the 3' exon. In this paper we report results from an in vitro analysis of wild-type, mutant, and pseudorevertant pre-mRNA splicing. We find that wild-type RNA is spliced inefficiently in vitro, and that the insertion mutation and suppressors act directly at the level of splicing. Characterization of splicing intermediates reveals that the insertion mutation and suppressor mutations located within the intron alter the pattern of lariat formation. In contrast, suppressor mutations consisting of 3' exon deletions act at an earlier step in the splicing pathway. Thus, the efficiency of splicing at the env 3' splice site can be affected at the level of spliceosome assembly, lariat formation, or cleavage at the 3' splice site and exon ligation.

Avian Sarcoma Viruses↗
PubMed1990

Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus.

A monoclonal antibody raised against mammalian spliceosomes specifically recognizes a non-snRNP factor required for spliceosome assembly. This splicing factor is highly concentrated in discrete regions within the nucleus, in a pattern that is a distinct subset of that seen with anti-snRNP antibodies. These observations are evidence that spliceosome assembly could be compartmentalized within the nucleus.

Animals↗
PubMed1988

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo.

The major nucleocapsid protein of avian retroviruses, pp 12, binds to single-stranded viral RNA with high affinity. Phosphorylation at Ser-40 is necessary for this binding. In order to examine the role of phosphorylation of serine 40 in the biological function of pp 12, we have introduced a series of amino acid substitutions at this position in the Rous sarcoma virus (Pr-C) protein. Substitution of threonine, alanine, or three other amino acids for Ser-40 had very little or no detectable effect on viral replication, nor did the control substitution of glycine for Ser-43, a nonphosphorylated residue. In vivo and in vitro, the Ala-40 and probably the Thr-40 substituted p 12 proteins are phosphorylated at alternative sites which are phosphorylated to a minor extent in vivo in the wild type protein. A study of the RNA binding properties of Ala-40 substituted p 12 has indicated that the protein has been stabilized in a high affinity RNA binding state which is independent of phosphorylation. The viability of the Ala-40 mutant virus indicates that this high binding affinity may be required for biological activity.

Amino Acid Sequence↗
PubMed1988

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12. Mutation which affects RNA binding in vitro blocks viral replication.

Site-directed mutagenesis was used to replace the lysine residues of the avian retrovirus nucleocapsid protein pp 12 that have previously been shown to be important for RNA binding. Single amino acid substitutions at Lys-36, -37, and -39 of the protein were not sufficient to affect virus production as measured by reverse transcriptase activity in virus particles released from transfected cells. However, when Lys-36 and Lys-37 were simultaneously replaced by isoleucine residues, there was a complete block of viral replication. As expected from the single mutant analyses, the wild type phenotype could be restored by reverting either or both of the isoleucine residues to lysine residues. Analysis of a bacterially produced rp 12 protein containing Ile-36 and Ile-37 indicated that the protein has a low affinity binding for RNA, as compared to wild type protein. Unlike wild type, the binding is independent of phosphorylation at Ser-40, the major site of phosphorylation of the protein in vivo. A quail cell line was established that expresses virus particles containing the doubly mutated pp 12. Analysis of these particles indicated that they lack viral RNA. Thus, the binding defect in pp 12 is correlated with the inability to package viral RNA.

Animals↗
PubMed1988

Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers".

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Cys. All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested experimentally. No tightly bound metal ions were detected for avian myeloblastosis nucleocapsid protein, and the molar amount of zinc in virions was less by a factor of 50 than that of the nucleocapsid protein. Added Zn2+ did not significantly affect nucleocapsid binding to poly(ethenoadenylic acid) or its secondary structure, as determined from circular dichroism. Nevertheless, the conserved cysteine and histidine residues of the Rous sarcoma (Prague-C strain) nucleocapsid protein are essential for fully functional virus, as shown by the fact that single-site substitutions of five of the six conserved cysteines and either of the two histidine residues blocked viral replication.

Avian Leukosis Virus↗