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Science (New York, N.Y.) Jan 2004RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of...
RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.
Topics: Amino Acid Sequence; Argonaute Proteins; Cell Cycle Proteins; Centromere; Chromosomes, Fungal; Endoribonucleases; Genes, Reporter; Heterochromatin; Mass Spectrometry; Models, Genetic; Molecular Sequence Data; Mutation; Precipitin Tests; Protein Binding; RNA Interference; RNA, Fungal; RNA, Small Interfering; RNA-Binding Proteins; Ribonuclease III; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 14704433
DOI: 10.1126/science.1093686 -
Current Biology : CB Jul 1999Replicative DNA polymerases generally cannot pass lesions in the template strand. Now there is accumulating evidence for the widespread existence of a separate class of...
Replicative DNA polymerases generally cannot pass lesions in the template strand. Now there is accumulating evidence for the widespread existence of a separate class of DNA polymerases that can carry out translesion synthesis in both mutagenic and error-free ways.
Topics: Bacterial Proteins; DNA; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Escherichia coli Proteins; Fungal Proteins; Humans; Mutation; Saccharomyces cerevisiae Proteins; Xeroderma Pigmentosum; DNA Polymerase iota
PubMed: 10395530
DOI: 10.1016/s0960-9822(99)80299-1 -
Nucleic Acids Research Jul 2021Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to...
Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.
Topics: Adenosine Triphosphatases; DNA, Fungal; DNA-Binding Proteins; Genomic Instability; Rad51 Recombinase; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Ubiquitin-Protein Ligases
PubMed: 34157114
DOI: 10.1093/nar/gkab511 -
The Journal of Biological Chemistry Oct 2001Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic...
Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER). We report here that a mammalian protein (Yip1A) that is about 31% identical to S. cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport. Yip1A transcripts are ubiquitously expressed. Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart. Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region. The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex. Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites. Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus. A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport. Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24. A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction. Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.
Topics: Amino Acid Sequence; Animals; CHO Cells; COP-Coated Vesicles; Carrier Proteins; Cell Compartmentation; Chlorocebus aethiops; Cricetinae; Endoplasmic Reticulum; Golgi Apparatus; HeLa Cells; Humans; Membrane Proteins; Mice; Molecular Sequence Data; Phosphoproteins; Protein Binding; Protein Transport; Proteins; Receptors, Peptide; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Vero Cells; Vesicular Transport Proteins
PubMed: 11489904
DOI: 10.1074/jbc.M106189200 -
Journal of Biochemistry Nov 1997The binding of nuclear envelope precursor vesicles and chromatin was characterized by using an in vitro system constituted from a Xenopus egg extract and demembranated...
The binding of nuclear envelope precursor vesicles and chromatin was characterized by using an in vitro system constituted from a Xenopus egg extract and demembranated Xenopus sperm chromatin. The results of binding studies in the presence of salts, urea, and a chelator showed that the binding involves an ionic interaction. Chemical modification studies suggested that a protein(s) in the vesicles, which is responsible for the binding with chromatin, has essential lysine, histidine, and methionine residues. The vesicle protein could not be extracted from vesicles with 1 M KCl, 2 M urea, or 0.1 M Na2CO3, suggesting that it is an intrinsic membrane protein. The protein was denatured with 8 M urea and 0.1 M Na2CO3, and could be renatured by incubation at 23 degrees C, suggesting that the native conformation of the protein is important for the binding. Affinity purification of nuclear envelope precursor vesicles was achieved by binding to chromatin and dissociation with 0.24 M NaCl. The vesicle fraction thus obtained exhibited the ability to form nuclear envelope on incubation with chromatin in Xenopus egg cytosol without any other membrane fraction. These results suggested that there is a nuclear envelope precursor vesicle population containing both a chromatin targeting protein and vesicle fusion machinery.
Topics: Animals; Centrifugation; Chemical Precipitation; Chromatin; Imidoesters; Male; Membrane Proteins; Nuclear Envelope; Protein Binding; Protein Precursors; Spermatozoa; Trypsin; Xenopus
PubMed: 9443820
DOI: 10.1093/oxfordjournals.jbchem.a021842 -
Journal of Biochemistry Feb 1995Plasma protein S is a cofactor of activated protein C (APC) in the regulation of the blood coagulation system. Rat protein S homogeneously purified from plasma showed... (Comparative Study)
Comparative Study
Plasma protein S is a cofactor of activated protein C (APC) in the regulation of the blood coagulation system. Rat protein S homogeneously purified from plasma showed cofactor activity for rat APC, but not for human APC when the APC cofactor activity was assayed using protein S- and C4b-binding protein (C4BP)-depleted human plasma. Rat plasma protein S was separated by gel chromatography into two forms, a free form and a form complexed with C4BP. Rat protein S forms complexes with rat and human C4BP in a solid-phase model with apparent dissociation constants (Kds) of 6.7 x 10(-8) and 1.2 x 10(-8) M, respectively, in the presence of 5 mM Ca2+. Human protein S also forms a complex with solid-phase human and rat C4BP with Kds of 6.3 x 10(-9) and 2.7 x 10(-8) M, respectively. Human C4BP strongly inhibited the APC cofactor activity of both human and rat protein S, whereas rat C4BP was only weakly inhibitory. The degree of the inhibitory activity of C4BP appears to depend on the affinity between protein S and C4BP. In order to evaluate the structure-function relationship of the rat protein S, the complete cDNA sequence of rat protein S was determined. This cDNA of 3,315 bp was composed of a 103-bp 5'-noncoding region, a 2,028-bp coding region that encodes a preprosequence of 41 amino acids, a mature protein S of 634 amino acids and a stop codon, and a 1,184-bp 3'-noncoding region. The rat mature protein S consisted of domains with distinct functions similar to those of human protein S, and with two potential Asn-linked glycosylation sites. The amino acid sequence of the mature form of rat protein S showed 80.4, 78.7, and 79.7% identity with those of human, bovine, and rabbit mature protein S, respectively. These findings suggest that despite the species-specificity of the APC cofactor activity of rat protein S, it is structurally very similar to human protein S. Expression of rat protein S mRNA (approximately 3.5 kb) was demonstrated by RNA blot analysis not only in the liver, but also in the lung, spleen, testis, and uterus of rats.
Topics: Animals; Base Sequence; Cattle; Cloning, Molecular; DNA Primers; DNA, Complementary; Gene Expression; Humans; Kinetics; Molecular Sequence Data; Partial Thromboplastin Time; Polymerase Chain Reaction; Protein C; Protein S; Rabbits; Rats; Recombinant Proteins; Restriction Mapping; Sequence Homology, Amino Acid
PubMed: 7608128
DOI: 10.1093/jb/117.2.374 -
Nature Communications Jun 2018Form and function of the mitotic spindle depend on motor proteins that crosslink microtubules and move them relative to each other. Among these are kinesin-14s, such as...
Form and function of the mitotic spindle depend on motor proteins that crosslink microtubules and move them relative to each other. Among these are kinesin-14s, such as Ncd, which interact with one microtubule via their non-processive motor domains and with another via their diffusive tail domains, the latter allowing the protein to slip along the microtubule surface. Little is known about the influence of the tail domains on the protein's performance. Here, we show that diffusive anchorage of Ncd's tail domains impacts velocity and force considerably. Tail domain slippage reduced velocities from 270 nm s to 60 nm s and forces from several piconewtons to the sub-piconewton range. These findings challenge the notion that kinesin-14 may act as an antagonizer of other crosslinking motors, such as kinesin-5, during mitosis. It rather suggests a role of kinesin-14 as a flexible element, pliantly sliding and crosslinking microtubules to facilitate remodeling of the mitotic spindle.
Topics: Drosophila Proteins; Green Fluorescent Proteins; Kinesins; Microtubule-Associated Proteins; Microtubules; Mitosis; Optical Tweezers; Protein Binding; Protein Domains; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 29880831
DOI: 10.1038/s41467-018-04656-0 -
RNA (New York, N.Y.) Feb 2003A critical step in the turnover of yeast mRNAs is decapping. Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in...
A critical step in the turnover of yeast mRNAs is decapping. Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established. To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties. These experiments demonstrate that copurification of Dcp1p and Dcp2p yields active decapping enzyme under a variety of conditions. Moreover, Dcp2p alone can have decapping activity under some biochemical conditions. This suggests that Dcp2p can be a catalytic subunit of the decapping complex, and Dcp1p may function to enhance Dcp2p activity, or as an additional active subunit. In addition, recombinant Dcp1p/Dcp2p prefers long mRNA substrates and is sensitive to inhibition by sequestration of the 5' end but not the 3' end of the substrate. This suggests that Dcp1p/Dcp2p contains an additional RNA-binding site spatially distinct from the active site. Finally, using two RNA-binding proteins that enhance decapping in vivo (Edc1p and Edc2p), we can reconstitute the activation of decapping with recombinant proteins. This indicates that the Edc1 and Edc2 proteins act directly on the decapping enzyme.
Topics: Endoribonucleases; RNA; RNA Cap-Binding Proteins; RNA Caps; RNA-Binding Proteins; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Yeasts
PubMed: 12554866
DOI: 10.1261/rna.2151403 -
Biochemical and Biophysical Research... Mar 2011Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the...
Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.
Topics: Circular Dichroism; Escherichia coli; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Protein Structure, Secondary; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 21295540
DOI: 10.1016/j.bbrc.2011.01.113 -
Biochimica Et Biophysica Acta Apr 2014Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both...
Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.
Topics: Bacterial Outer Membrane Proteins; DNA, Fungal; Phosphotransferases (Phosphate Group Acceptor); Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; RNA Splicing Factors; RNA, Fungal; RNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 24382491
DOI: 10.1016/j.bbapap.2013.12.016