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PubMed2002

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein.

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

Chitin↗
PubMed2005

Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo.

Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein-protein as well as protein-RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome.

Alternative Splicing↗
PubMed2002

The SR protein SRp38 represses splicing in M phase cells.

SR proteins constitute a family of pre-mRNA splicing factors that play important roles in both constitutive and regulated splicing. Here, we describe one member of the family, which we call SRp38, with unexpected properties. Unlike other SR proteins, SRp38 cannot activate splicing and is essentially inactive in splicing assays. However, dephosphorylation converts SRp38 to a potent, general repressor that inhibits splicing at an early step. To investigate the cellular function of SRp38, we examined its possible role in cell cycle control. We show first that splicing, like other steps in gene expression, is inhibited in extracts of mitotic cells. Strikingly, SRp38 was found to be dephosphorylated specifically in mitotic cells, and we show that dephosphorylated SRp38 is required for the observed splicing repression.

Alternative Splicing↗
PubMed1998

A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy.

Pre-mRNA splicing is an important regulatory step in the expression of most eukaryotic genes. In vitro studies have shown splicing to occur within 50-60 S multi-component ribonucleoprotein (RNP) complexes termed spliceosomes. Studies of mammalian cell nuclei have revealed larger complexes that sediment at 200 S in sucrose gradients, termed large nuclear RNP (lnRNP) particles. These particles contain all factors required for pre-mRNA splicing, including the spliceosomal U snRNPs and protein splicing factors. Electron microscopy has shown them to consist of four apparently similar substructures. In this study, mass measurements by scanning transmission electron microscopy of freeze-dried mammalian lnRNP preparations, both confirm the similarity between the lnRNP particles and reveal the mass uniformity of their subunits. Thus, the tetrameric lnRNP particle has a mass of 21.1(+/-1.6) MDa, while each repeating subunit has a mass of 4.8(+/-0.5) MDa, which is close to the estimated mass of the fully assembled 60 S spliceosome. The 1.9 MDa discrepancy between the lnRNP particle's mass and the cumulative masses of its four subunits may be attributed to an additional domain frequently observed in the micrographs. Notably, strands and loops of RNA were often seen emanating from lnRNP particles positively stained with uranyl formate. Our results support the idea that the nuclear splicing machine is a supraspliceosome complex. For clarity, we define spliceosomes devoid of pre-mRNA as spliceosome cores, and propose that the supraspliceosome is constructed from one pre-mRNA, four spliceosome cores, each composed mainly of U snRNPs, and additional proteins. In this way a frame is provided to juxtapose exons about to be spliced.

Mass Spectrometry↗
PubMed1991

The TPR snap helix: a novel protein repeat motif from mitosis to transcription.

The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi-domain proteins all contain tandemly arranged repeats of a 34-amino acid motif that are presumed to form helix-turn structures, each with a 'knob' and 'hole', acting as helix-associating domains.

Amino Acid Sequence↗
PubMed1998

Structural similarities between hammerhead ribozymes and the spliceosomal RNAs could be responsible for lack of ribozyme cleavage in yeast.

Hammerhead ribozymes have been proposed as potential therapeutic agents for the treatment of viral and other diseases. However, a clear understanding of the cleavage reaction in vivo is not available at present. In these studies, we chose the yeast Saccharomyces cerevisiae as a model system to study the effects of hammerhead cleavage on gene expression in vivo. Several reporter genes were employed to monitor the self-cleaving characteristics of three different ribozymes. We show that these ribozymes decrease expression of some reporter genes by interfering with splice site selection or translation initiation and not by in vivo cleavage of the RNA transcripts. In fact, it appears that although these ribozymes can efficiently self-cleave the RNA in vitro, they are not able to function in vivo. We have identified a yeast splicing protein that interacts in vivo with our cis-ribozyme by specifically recognizing the ribozyme structure (Lin and Rossi, 1996). This interaction does not occur if different secondary structures are used in place of the ribozyme. The binding of this protein to the ribozyme can account for the inability of ribozymes to efficiently cleave in yeast. Remarkably, when yeast extracts are added to in vitro trans-cleavage reactions, the cleavage ability of the ribozyme is hampered, whereas the addition of mammalian extracts yields an enhancement of the reactions. These results confirm the presence of factor(s) that can block ribozyme function in the yeast intracellular environment.

Base Sequence↗
PubMed1999

Golli-induced paralysis: a study in anergy and disease.

The Golli-MBP transcription unit contains three Golli-specific exons as well as the seven exons of the classical myelin basic protein (MBP) gene and encodes alternatively spliced proteins that share amino acid sequence with MBP. Unlike MBP, which is a late Ag expressed only in the nervous system, Golli exon-containing gene products are expressed both pre- and postnatally at many sites, including lymphoid tissue, as well as in the central nervous system. To investigate whether Golli-MBP peptides unique to Golli would result in neurological disease, we immunized rats and observed a novel neurological disease characterized by mild paralysis and the presence of groups of lymphocytes in the subarachnoid space but not in the parenchyma of the brain. Disease was induced by Th1-type T cells that displayed an unusual activation phenotype. Primary stimulation in vitro induced T cell proliferation with increased surface CD45RC that did not become down-regulated as it did in other Ag-stimulated cultures. Secondary stimulation of this CD45RChigh population with Ag, however, did not induce proliferation or IL-2 production, although an IFN-gamma-producing population resulted. Proliferation could be induced by secondary stimulation with IL-2 or PMA-ionomycin, suggesting an anergic T cell population. Cells could adoptively transfer disease after secondary stimulation with IL-2, but not with Ag alone. These responses are suggestive of a chronically stimulated, anergic population that can be transiently activated to cause disease, fall back into an anergic state, and reactivated to cause disease again. Such a scenario may be important in chronic human disease.

Adoptive Transfer↗
PubMed2006

Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.

High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.

Aminopeptidases↗
PubMed1999

Improved segmental isotope labeling of proteins and application to a larger protein.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

ATP-Binding Cassette Transporters↗
PubMed2005

Development of a tandem protein trans-splicing system based on native and engineered split inteins.

Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chemical biology and biotechnology, precious little is known about the molecular recognition events governing the initial fragment association step. In this study, fluorescence approaches have been used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid association of the fragments at physiological pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technology should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins.

Amino Acid Sequence↗
PubMed2005

Genetically encoded stress indicator for noninvasively imaging endogenous corticosterone in living mice.

Physical and emotional stress is one of the major controllers of physiological reactions and homeostasis in living animals. A stress hormone, corticosterone, is secreted from the adrenal cortex into the blood vessels when animals sense the stress. The quantitative evaluation of corticosterone in living animals has been limited because of the unavailability of suitable methods in vivo. For a noninvasive molecular imaging of the stress, we developed a method for detecting physiological increases in the endogenous corticosterone caused by exo- and endogenous stress in living animals. We constructed a pair of genetically encoded indicators composed of cDNAs of glucocorticoid receptor (GR), split Renilla luciferase (RLuc), and a Synechocystis sp. DnaE intein. The GR fused with C-terminal halves of RLuc and DnaE is localized in the cytosol, whereas a fusion protein of N-terminal halves of RLuc and DnaE is localized in the nucleus. If corticosterone induces GR translocation into the nucleus, the C-terminal RLuc meets the N-terminal one in the nucleus, and full-length RLuc is reconstituted by protein splicing with DnaE. Cell-based methods provided a quantitative bioluminescence assay of the extent of GR translocation into the nucleus. We further demonstrated that the indicator enabled noninvasive imaging against two different types of imposed stress: a forced swimming and metabolic perturbation caused by 2-deoxy-D-glucose. This stress indicator should be valuable for screening pharmacological compounds and for tools to study the mechanism of physiological stress.

Animals↗
PubMed2006

The nuclear-encoded inteins of fungi.

An intein is a protein sequence embedded within a precursor protein that is excised during protein maturation. Inteins were first found encoded in the VMA gene of Saccharomyces cerevisiae. Subsequently, they have been found in diverse organisms (eukaryotes, archaea, eubacteria and viruses). The VMA intein has been found in various saccharomycete yeasts but not in other fungi. Different inteins have now been found widely in the fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins. A protein distantly related to inteins, but closely related to metazoan hedgehog proteins, has been described from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active. The enlarged fungal intein data set permits insight into the evolution of inteins, including the role of horizontal transfer in their persistence. The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.

Amino Acid Sequence↗
PubMed1999

Intein-mediated protein ligation: harnessing nature's escape artists.

Inteins are naturally occurring proteins that are involved in the precise cleavage and formation of peptide bonds in a process known as protein splicing. Genetic engineering has allowed the controllable cleavage of peptide bonds at either the N- or C-terminus of the intein. Inteins displaying controllable cleavage have been used in the isolation of bacterially expressed proteins possessing either a C-terminal thioester or an N-terminal cysteine. The specific placement of these reactive groups has allowed either protein-protein or protein-peptide condensation through a native peptide bond. This review describes the methods used to specifically generate these reactive groups on bacterially expressed proteins and some applications of this technique, known as intein-mediated protein ligation. Furthermore, a versatile two intein (TWIN) system will be described which enables the circularization and polymerization of bacterially expressed proteins or peptides.

Amino Acid Motifs↗
PubMed1997

A DnaB intein in Rhodothermus marinus: indication of recent intein homing across remotely related organisms.

A dnaB gene encoding a homologue of the Escherichia coli DNA helicase DnaB was cloned and sequenced in the thermophilic eubacterium Rhodothermus marinus, predicting a DnaB protein that harbors an intein. This DnaB intein is 428 amino acid residues long, has several putative intein sequence motifs (including two putative endonuclease motifs), and is capable of protein splicing when produced in E. coli cells. The R. marinus DnaB intein is a close homologue of a DnaB intein in the cyanobacterium Synechocystis sp. strain PCC6803. The two inteins are positioned identically in their respective DnaB proteins. They also share a 54% sequence identity (74% sequence similarity) that is markedly higher than the 37% sequence identity shared by the extein sequences of the two DnaB proteins. Horizontal intein transfer (homing) is therefore invoked to relate these two DnaB inteins. The codon usage of R. marinus DnaB intein coding sequence differs markedly from the codon usages of its flanking extein coding sequences and other genes in the same genome, suggesting more recent acquisition of the DnaB intein in this organism.

Amino Acid Sequence↗
PubMed1998

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii.

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.

Amino Acid Sequence↗
PubMed2006

Studies on the role of NonA in mRNA biogenesis.

The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A)(+) RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles.

Active Transport, Cell Nucleus↗
PubMed2000

Molecular basis for modulation of biological function by alternate splicing of the Wilms' tumor suppressor protein.

Alternate splicing, leading to the insertion of the tripeptide KTS in the linker between the third and fourth C(2)H(2) zinc fingers, changes both the DNA-binding function and the subnuclear localization of the Wilms' tumor suppressor protein (WT1). We have used NMR relaxation experiments to determine the molecular basis for the differing DNA recognition properties of the WT1-KTS and WT1+KTS isoforms. Our results show that the KTS insertion increases the flexibility of the linker between fingers 3 and 4 and abrogates binding of the fourth zinc finger to its cognate site in the DNA major groove. This represents a mechanism whereby a single zinc-finger gene can be used, through alternate splicing, to fulfill different functions in the cell.

Alternative Splicing↗