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PubMed2005

Low temperature promotes intron retention in two e-cor genes of durum wheat.

Following the screening of a suppression subtractive library developed from durum wheat plants exposed to low temperature for 6 h, two early cold-regulated (e-cor) genes have been isolated. These genes, coding putatively for a ribokinase (7H8) and a C3H2C3 RING-finger protein (6G2), were characterized by the stress-induced retention of a subset of introns in the mature mRNA. This feature was dependent on cold for 7H8 and on cold and dehydration for 6G2. When other genes, such as the stress-related gene WCOR410c, coding for a dehydrin (one intron), or a gene coding for a putative ATP binding cassette transporter (16 introns) were analyzed, no cold-dependent intron retention was observed. Cold-induced intron retention was not observed in mutants defective in the chloroplast development; nevertheless treatment with cycloheximide in the absence of cold was able to promote intron retention for the 7H8 e-cor gene. These results suggest that the cold-induced intron retention reflects the response of the spliceosoma to specific environmental signals transduced to the splicing protein factors through a chloroplast-dependent pathway. Notably, when the 7H8 Arabidopsis orthologous gene was analyzed, no stress induction in terms of mRNA abundance and no cold-dependent intron retention was detected. Otherwise, 6G2 Arabidopsis homologous sequences sharing the same genomic structure of the durum wheat 6G2 showed a similar intron retention event although not strictly dependent on stress.

Arabidopsis↗
PubMed2005

Structural analysis and expression profile of a novel gene on chromosome 5q23 encoding a Golgi-associated protein with six splice variants, and involved within the 5q deletion of a Ph(-) CML patient.

We have identified a novel gene, upstream of the cytokine gene cluster region in 5q23-31, residing within one of the most common deleted segments associated with MDS. The novel gene exhibits significant alternative splicing generating at least six splice variants encoding four putative proline-rich protein isoforms, one of which is Golgi-associated. The gene is ubiquitously expressed and conserved among species with the C. elegans homologue being the most interesting, since it resides within an operon with two other genes, phospholipase D and dishevelled, a member of the Wnt pathway, suggesting a functional association. In addition, the novel gene and other key regulatory genes of the region, such IL3, Ril, AF5q31 and TCF-1, were found to be deleted in an atypical CML case, thus underscoring the significance of this subregion in the leukemogenesis process.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Fmoc-based synthesis of peptide alpha-thioesters using an aryl hydrazine support.

C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.

Journal Article↗
PubMed1999

Crystallization and preliminary X-ray analysis of a new L-aminopeptidase-D-amidase/D-esterase activated by a Gly-Ser peptide bond hydrolysis.

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group presumably used as a general base in the catalytic mechanism. Two crystal forms were obtained at 294 K in 13-16% PEG 2000 mono-methylether at pH 9.0, adding either 0.2 M magnesium chloride or 1 M lithium chloride. Crystals of the first form belong to the space group C2221 and diffract to 3.0 A resolution, whereas crystals of the second form belong to the space group P21212 and diffract to 2.3 A resolution. Initial screening for heavy-atom derivatives on form II crystals, has led to a well substituted Hg derivative.

Aminopeptidases↗
PubMed2003

Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA.

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.

Adenosine Triphosphate↗
PubMed1996

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Amino Acid Sequence↗
PubMed1993

Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing.

The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification; however, we cannot eliminate the possibility that a small fraction of PrPSc is modified by an as yet unidentified posttranslational process or that PrPC carries a modification that is removed in the formation of PrPSc. It seems likely that PrPSc differs from PrPC in its secondary and tertiary structure, but the possibility of a tightly bound, disease-specific molecule which purifies with PrPSc must also be considered.

Amino Acid Sequence↗
PubMed2005

A new example of viral intein in Mimivirus.

BACKGROUND: Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in all domains of life, but only once to date in the genome of a eukaryote-infecting virus. RESULTS: Here we report the identification and bioinformatics characterization of an intein in the DNA polymerase PolB gene of amoeba infecting Mimivirus, the largest known double-stranded DNA virus, the origin of which has been proposed to predate the emergence of eukaryotes. Mimivirus intein exhibits canonical sequence motifs and clearly belongs to a subclass of archaeal inteins always found in the same location of PolB genes. On the other hand, the Mimivirus PolB is most similar to eukaryotic Poldelta sequences. CONCLUSIONS: The intriguing association of an extremophilic archaeal-type intein with a mesophilic eukaryotic-like PolB in Mimivirus is consistent with the hypothesis that DNA viruses might have been the central reservoir of inteins throughout the course of evolution.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Base Sequence↗
PubMed2002

Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity.

Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the protein-splicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single- and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both Mn(2+) and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel Mn(2+)-ATP-dependent double-strand specific endonuclease, which is likely to be important for homing process in vivo.

Adenosine Triphosphate↗
PubMed1999

Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence.

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.

Base Sequence↗
PubMed2002

Multiple roles for SR proteins in trans splicing.

The trans-splicing reaction involves the association of 5' and 3' splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3' acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5' splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.

Animals↗
PubMed2003

Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with concomitant joining of the flanking sequences. Here, we demonstrate intein trans-splicing in Nicotiana tabacum chloroplasts by using the naturally split Ssp DnaE intein. Trans-splicing occurred whether both intein fragments were encoded in the chloroplast or were separated into the chloroplast and nuclear genomes. A biolistic approach was used to integrate two fusion genes, one encoding aminoglycoside-3-adenyltransferase (aadA) and the first 123 aa of the Ssp DnaE intein (In) and the other encoding 36 C-terminal amino acid residues of the Ssp DnaE intein (Ic) and soluble modified green fluorescent protein (smGFP) into N. tabacum plastids. Expression of these gene fragments in the chloroplast resulted in ligated aadA-smGFP due to In-Ic-mediated trans-splicing. Furthermore, an N-terminal portion of the herbicide resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) containing a chloroplast localization signal fused to In (EPSPSn-In) was integrated into the nuclear DNA of N. tabacum by using Agrobacterium tumefaciens-mediated transformation. The remaining EPSPS gene fragment (EPSPSc) fused to Ic (Ic-EPSPSc) was integrated into the chloroplast genome by homologous recombination. Western blot analysis of cell extracts from these plants showed a full-length EPSPS, demonstrating that the EPSPSn-In gene product migrated to the chloroplast and underwent trans-splicing. Furthermore, these transgenic plants displayed improved resistance to the herbicide N-(phosphonomethyl)glycine (glyphosate) when compared with wild-type N. tabacum.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2024

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7&#x2009;kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14&#x2009;kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2&#x2009;&#xd7;&#x2009;1013&#x2009;viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that&#xa0;exceed the AAV capacity.

Animals↗
PubMed1999

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Amino Acid Sequence↗
PubMed1993

The spliceosome.

The spliceosome is a large RNA-protein complex that catalyses the removal of introns from nuclear pre-mRNA. A wide range of biochemical and genetical studies shows that the spliceosome comprises three major RNA-protein subunits, the U1, U2 and [U4/U6.U5] small nuclear ribonucleoprotein particles (snRNPs), and an additional group of non-snRNP protein splicing factors. Rapid progress is being made in unravelling the interactions which take place between these factors during the splicing reaction. The emerging picture of the spliceosome reveals a highly dynamic structure that assembles on pre-mRNA transcripts in a stepwise pathway and is organised, at least in part, by complex RNA base-pairing interactions between the small nuclear RNAs (snRNAs) and the intron substrate. Many of these interactions can be detected both in mammalian and yeast spliceosomes, suggesting that the basic splicing mechanism is an ancient one that has been highly conserved during evolution.

Base Composition↗
PubMed1994

Phosphoribosylpyrophosphate synthetase (PRS): a new gene family in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains at least four PRS genes, all of which have been cloned and sequenced. Each of the four derived amino acid sequences have more than 60% similarity to the corresponding polypeptides of man, rat, Escherichia coli and Salmonella typhimurium. The PRS1 gene maps on chromosome XI, PRS2 on chromosome V, PRS3 on chromosome VIII and PRS4 on chromosome II. One member of this gene family, PRS1, contains a region of non-homology (NHR) shown by cDNA cloning and sequencing not to be an intron. The results presented here suggest that the presence of this NHR is not detrimental to the function of the gene. To date the possibility of protein splicing can be neither proven nor disputed.

Amino Acid Sequence↗