PRP8 inteins in species of the genus Botrytis and other ascomycetes

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.     

Intein harbouring large tandem repeats in replicative DNA helicase of Trichodesmium erythraeum

Protein splicing inteins can be small as approximately 130 aa or up to approximately 600 aa when harbouring an endonuclease domain. Here we report the identification and characterization of an unusually large intein, 1650 aa long and the largest of known inteins, encoded by the replicative DNA helicase gene dnaB of the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. This Ter DnaB-1 intein co-exists with a 177-aa mini-intein in the same host protein and harbours large tandem repeats in which an 84-aa sequence is repeated 16 times. Comparison between this tandem repeats and the recently reported tandem repeats of Ter DnaE-1 intein revealed differences and similarities. The two tandem repeats, residing in different inteins of different host proteins, differ by 50% in size and have little sequence similarity. Tandem repeats in the Ter DnaB-1 intein were required for the protein splicing activity when tested in Escherichia coli, in contrast to tandem repeats of the Ter DnaE-1 intein that inhibited protein splicing. On the other hand, tandem repeats of both inteins are located in the same corresponding region of the intein sequence and have the same number of repeating units. These suggest that the two tandem repeats could be related but have diverged greatly in size, sequence and effect on protein splicing. Alternatively, they could have independent origins but evolved certain similarities because of common constraints in structure and maintenance.     

The PRP8 inteins in Cryptococcus are a source of phylogenetic and epidemiological information

Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.     

Evaluation and comparison of protein splicing by exogenous inteins with foreign exteins in Escherichia coli

Protein splicing catalyzed by inteins has enabled various biotechnological applications such as protein ligation. Successful applications of inteins are often limited by splicing efficiency. Here, we report the comparison of protein splicing between 20 different inteins from various organisms in identical contexts to identify robust inteins with foreign exteins. We found that RadA intein from Pyrococcus horikoshii and an engineered DnaB intein from Nostoc punctiforme demonstrated an equally efficient splicing activity to the previously reported highly efficient DnaE intein from Nostoc punctiforme. The newly identified inteins with efficient cis-splicing activity can be good starting points for the further development of new protein engineering tools.     

Split dnaE genes encoding multiple novel inteins in Trichodesmium erythraeum

Three inteins were found when analyzing a pair of split dnaE genes encoding the catalytic subunit of DNA polymerase III in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The three inteins (DnaE-1, DnaE-2, and DnaE-3) were clustered in a 70-amino acid (aa) region of the predicted DnaE protein. The DnaE-1 intein is 1258 aa long and three times as large as a typical intein, due to the presence of large tandem repeats in which a 57-aa sequence is repeated 17 times. The DnaE-2 intein has a more typical size of 428 aa with putative protein splicing and endonuclease domains. The DnaE-3 intein is a split intein consisting of a 102-aa N-terminal part and a 36-aa C-terminal part encoded on the first and second split dnaE genes, respectively. Synthesis of a mature DnaE protein is predicted to involve expression of two split dnaE genes followed by two protein cis-splicing reactions and one protein trans-splicing reaction. Tandem repeats in the DnaE-1 intein inhibited the protein splicing activity of this intein when tested in Escherichia coli cells and may potentially regulate DnaE synthesis in vivo.     

Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein

As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.     

Compilation and analysis of intein sequences.

We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996. Analysis of the 36 available intein sequences refines the previously described intein motifs and reveals the presence of another intein motif, Block H. Furthermore, analysis of the new inteins reshapes our view of the conserved splice junction residues, since three inteins lack the intein penultimate His seen in prior examples. Comparison of intein sequences suggests that, in general, (i) inteins present in the same location within extein homologs from different organisms are very closely related to each other in paired sequence comparison or phylogenetic analysis and we suggest that they should be considered intein alleles; (ii) multiple inteins present in the same gene are no more similar to each other than to inteins present in different genes; (iii) phylogenetic analysis indicates that inteins are so divergent that trees with statistically significant branches cannot be generated except for intein alleles.     

Sexual mating of Botrytis cinerea illustrates PRP8 intein HEG activity

Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/?). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/? heterozygote, the homing endonuclease initiates intein ‘homing’ by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.     

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.     

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.     

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.     

Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ～2 μM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ～40 μM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.     

Modular organization of inteins and C-terminal autocatalytic domains.

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.     

Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution

Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an ～60-fold increased rate in the protein trans-splicing reaction and a K(d) value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed.     

Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity

Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes.     

多种S1断裂内含肽的体内交叉反应

断裂内含肽含有两个独立分离的多肽片段(N端内含肽和C端内含肽),它催化蛋白质反式剪接反应,在蛋白质研究与蛋白质工程中已得到诸多实际应用.在蛋白质反式剪接过程中,内含肽的N端内含肽和C端内含肽通过结构互补特异性地非共价组合.然而,Ssp DnaX S1型断裂内含肽的较大C端内含肽片段近来被发现能够与源自其它内含肽的N端内含肽片段交叉反应,表明蛋白质内含子Ssp DnaX具有结构杂交特征.本研究对另外2种S1型内含肽Rma DnaB和Ssp GyrB的较大C端内含肽与不同S1型断裂内含肽的N 端内含肽交叉反应活性进行分析检测.目的是探讨S1型断裂内含肽的结构杂交特征是否具有普遍性.结果发现,Rma DnaB的S1 C端内含肽能够与Ssp GyrB的S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应;与此相似,Ssp GyrB的S1 C端内含肽能够与Rma DnaB的 S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应.此外,某些交叉反应表现出温度依赖性.这些结果对于内含肽的结构 功能关系以及S1型断裂内含肽的应用研究具有重要的意义.     

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.     

Development of a positive genetic selection system for inhibition of protein splicing using mycobacterial inteins in Escherichia coli DNA gyrase subunit A

An intein-based positive genetic selection system was developed to study protein splicing and to provide a selection system with the potential for finding splicing inhibitors. Inteins can be novel antimicrobial targets when present in essential proteins since blocking splicing would kill the organism. For example, pathogenic mycobacteria encode inteins that interrupt DNA gyrase. The gyrase selection system exploits (1) splicing of inteins out of Gyrase A and (2) the dominant lethal effect of quinolone poisoning of DNA gyrase, which in turn blocks replication. The system was adapted for whole-cell high-throughput screening using green fluorescent protein as an automatable readout of viability. To demonstrate the efficacy of this system, mutations that blocked splicing of the Mycobacterium xenopi Gyrase A intein were isolated. Splicing was then assayed at a second temperature to identify inteins with a temperature-sensitive splicing phenotype. Mutations were mapped onto a structure-based sequence alignment, which led to the rational prediction of a temperature-sensitive splicing mutation. GyrA intein subdomain relationships also provided insight into intein evolution.     

Intein spread and extinction in evolution

Inteins are selfish DNA elements found within coding regions. They are translated with their host protein, but then catalyze their own excision and the formation of a peptide bond between their flanking protein regions. Understanding what drives and selects inteins is relevant for assessing whether they have unidentified biological functions and whether they can invade and become established in new genes and organisms. Inteins are suggested to have been present and more common in the progenitors of eukaryotes and prokaryotes. In these cells, inteins had some beneficial function or had evolved from an unknown beneficial protein. Since then, this putative benefit has been lost and inteins are gradually becoming extinct. The proteins in which inteins are currently found are proposed to be proteins vital for the survival of the organism, where intein removal is most difficult.