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At least 289 records · Page 16Linked to original sources
PubMed2000

TLS-ERG leukemia fusion protein inhibits RNA splicing mediated by serine-arginine proteins.

The translocation liposarcoma (TLS) gene is fused to the ETS-related gene (ERG) in human myeloid leukemia, resulting in the generation of a TLS-ERG protein. We demonstrate that both TLS and the TLS-ERG leukemia fusion protein bind to RNA polymerase II through the TLS N-terminal domain, which is retained in the fusion protein; however, TLS recruits members of the serine-arginine (SR) family of splicing factors through its C-terminal domain, whereas the TLS-ERG fusion protein lacks the ability to recruit SR proteins due to replacement of the C-terminal domain by the fusion partner ERG. In transient-transfection assays, the TLS-ERG fusion protein inhibits E1A pre-mRNA splicing mediated by these TLS-associated SR proteins (TASR), and stable expression of the TLS-ERG fusion protein in K562 cells alters the splicing profile of CD44 mRNA. These results suggest that TLS fusion proteins may lead to cellular abnormalities by interfering with the splicing of important cellular regulators.

Adenovirus E1A Proteins↗
PubMed1991

Sulfatide activator protein. Alternative splicing that generates three mRNAs and a newly found mutation responsible for a clinical disease.

The sulfatide activator protein, also known as SAP-1, is derived from a gene that generates an mRNA coding for four homologous proteins. Its physiological function is to stimulate hydrolysis of sulfatide by arylsulfatase A in vivo. A genetic defect in the sulfatide activator results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is itself caused by a genetic defect in arylsulfatase A. In a patient with sulfatide activator deficiency, a nucleotide transversion G722----C (counted from A of the initiation codon ATG) was found in the mRNA of the sulfatide activator precursor, resulting in the substitution of serine for Cys241 in the mature sulfatide activator. The remainder of the coding sequence was completely normal except for a polymorphism C to T in position 1389, which does not change the amino acid sequence. The patient produces at least three different forms of mRNA for the precursor. Two of them include a stretch of an additional 9 and 6 bases, respectively, within the sulfatide activator coding region. In normal individuals this stretch of additional bases has also been observed. This could be explained by the presence of a small 9-base pair exon which can be introduced, or not, by alternative splicing as a stretch of 9 or 6 bases into the mature mRNA. The shortest form of the mRNA yields an active sulfatide activator (Fürst, W., Schubert, J., Machleidt, W., Meier, H. E., and Sandhoff, K. (1990) Eur. J. Biochem. 192, 709-714).

Base Sequence↗
PubMed2004

Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA.

The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3' truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.

Amino Acid Sequence↗
PubMed1999

Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.

Alanine↗
PubMed2004

Synthetic two-piece and three-piece split inteins for protein trans-splicing.

Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do trans-splicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between beta-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar beta-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.

Escherichia coli↗
PubMed1989

Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria.

We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.

Amino Acid Sequence↗
PubMed2003

Four inteins and three group II introns encoded in a bacterial ribonucleotide reductase gene.

A bacterial ribonucleotide reductase gene was found to encode four inteins and three group II introns in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The 13,650-bp ribonucleotide reductase gene is divided into eight extein- or exon-coding sequences that together encode a 768-amino acid mature ribonucleotide reductase protein, with 83% of the gene sequence encoding introns and inteins. The four inteins are encoded on the second half of the gene, and each has conserved sequence motifs for a protein-splicing domain and an endonuclease domain. These four inteins, together with known inteins, define five intein insertion sites in ribonucleotide reductase homologues. Two of the insertion sites are 10 amino acids apart and next to key catalytic residues of the enzyme. Protein-splicing activities of all four inteins were demonstrated in Escherichia coli. The four inteins coexist with three group II introns encoded on the first half of the same gene, which suggests a breakdown of the presumed barrier against intron insertion in this bacterial conserved protein-coding gene.

Amino Acid Motifs↗
PubMed1994

SWAP pre-mRNA splicing regulators are a novel, ancient protein family sharing a highly conserved sequence motif with the prp21 family of constitutive splicing proteins.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.

Animals↗
PubMed2005

Protein posttranslational modifications: the chemistry of proteome diversifications.

The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.

Animals↗
PubMed2001

High yield expression and single step purification of human thionein/metallothionein.

Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purification with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd(7)- or Zn(7)-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus.

Amino Acid Sequence↗
PubMed2006

Analysis of the ribosomal protein S19 interactome.

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Glutathione Transferase↗
PubMed2002

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA.

Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclear-encoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA. The active six-subunit complex has a molecular mass of 420 kDa, splices with a k(cat) of 0.3 min(-1), and binds the guanosine nucleophile with an affinity comparable to other group I introns. The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNA-binding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity. Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding. Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions.

Amino Acid Sequence↗
PubMed2006

Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme.

Protein trans-splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non-native exteins in Escherichia coli. Npu DnaE possesses robust trans-splicing activity with an efficiency of > 98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N- and C-terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans-splicing activity. Protein splicing with the Npu DnaEN is also more tolerant of amino acid substitutions in the C-terminal extein sequence.

Amino Acid Sequence↗
PubMed1998

Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein.

A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp. strain PCC6803. This intein is shown to be capable of protein splicing with or without its native exteins when tested in E. coli cells. A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size. Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G. These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein.

Amino Acid Sequence↗
PubMed2001

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.

Base Sequence↗
PubMed2002

High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI.

The homing endonuclease PI-SceI from Saccharo myces cerevisiae consists of two domains. The protein splicing domain I catalyzes the excision of the mature endonuclease (intein) from a precursor protein and the religation of the flanking amino acid sequences (exteins) to a functional protein. Furthermore, domain I is involved in binding and recognition of the specific DNA substrate. Domain II of PI-SceI, the endonuclease domain, which is structurally homologous to other homing endonucleases from the LAGLIDADG family, harbors the endonucleolytic center of PI-SceI, which in vivo initiates the homing process by introducing a double-strand cut in the approximately 35 bp recognition sequence. At 1.35 A resolution, the crystal structure of PI-SceI domain I provides a detailed view of the part of the protein that is responsible for tight and specific DNA binding. A geometry-based docking of the 75 degrees bent recognition sequence to the full-length protein implies a conformational change or hinge movement of a subdomain of domain I, the tongs part, that is predicted to reach into the major groove near base pairs +16 to +18.

Binding Sites↗
PubMed1995

Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron.

The splicing factor CBP2 is required to excise the yeast mitochondrial group I intron bI5 in vivo and at low magnesium ion concentrations in vitro. CBP2 binding is strengthened 20-fold by increasing Mg2+ concentrations from 5 to 40 mM, implying the protein binds, in part, to the same structure as that stabilized by the cation. The same transition is also observed as a cooperative increase in the rate of self-processing between 5 and 40 mM Mg2+, providing strong evidence for an RNA folding transition promoted by either Mg2+ or CBP2. The first step of splicing, guanosine addition at the 5' splice site, is rate limiting for exon ligation. At low (5 mM) magnesium ion, reaction (measured as kcat/Km or kcat) is accelerated 3 orders of magnitude by saturating CBP2. At near-saturating Mg2+ (40 mM), acceleration is 8- and 30-fold, for kcat and kcat/Km, respectively, so high magnesium ion concentrations fail to compensate completely for protein facilitation. Thus, self-splicing proceeds via two additional transitions as compared with reaction of the bI5-CBP2 complex, only the first of which is efficiently promoted by the cation. Guanosine 5'-monophosphate binds (Kd approximately 0.3 mM) with the same affinity to bI5 and the bI5-protein complex, supporting independent binding of the nucleophile and CBP2. Substitution of a phosphorothioate at the 5' splice site and pH profiles provide evidence that kcat is limited by chemistry at low pH and by a conformational step at high pH. Because binding by either Mg2+ or CBP2 increases the rate of chemistry more than the rate of the conformational step, in the physiological pH range (7-7.6) the protein-facilitated reaction is limited by a conformation step while self-splicing reaction is limited by chemistry. We conclude that CBP2 makes manifold contributions to bI5 splicing: binding compensates for at least two structural defects and accelerates the rate of the chemistry.

Base Sequence↗