An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.     

Splice junctions are constrained by protein disorder

We have discovered that positions of splice junctions in genes are constrained by the tolerance for disorder-promoting amino acids in the translated protein region. It is known that efficient splicing requires nucleotide bias at the splice junction; the preferred usage produces a distribution of amino acids that is disorder-promoting. We observe that efficiency of splicing, as seen in the amino-acid distribution, is not compromised to accommodate globular structure. Thus we infer that it is the positions of splice junctions in the gene that must be under constraint by the local protein environment. Examining exonic splicing enhancers found near the splice junction in the gene, reveals that these (short DNA motifs) are more prevalent in exons that encode disordered protein regions than exons encoding structured regions. Thus we also conclude that local protein features constrain efficient splicing more in structure than in disorder.     

Probing intein-catalyzed thioester formation by unnatural amino acid substitutions in the active site

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers β-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.     

Splicing of the Mycobacteriophage Bethlehem DnaB Intein: IDENTIFICATION OF A NEW MECHANISTIC CLASS OF INTEINS THAT CONTAIN AN OBLIGATE BLOCK F NUCLEOPHILE*?

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala¹ inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.     

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

Control of protein splicing by intein fragment reassembly.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.     

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.     

Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*

Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves. N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.     

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.     

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.     

RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression

A systematic analysis of the RNA splice junction sequences of eukaryotic protein coding genes was carried out using the GENBANK databank. Nucleotide frequencies obtained for the highly conserved regions around the splice sites for different categories of organisms closely agree with each other. A striking similarity among the rare splice junctions which do not contain AG at the 3' splice site or GT at the 5' splice site indicates the existence of special mechanisms to recognize them, and that these unique signals may be involved in crucial gene-regulation events and in differentiation. A method was developed to predict potential exons in a bare sequence, using a scoring and ranking scheme based on nucleotide weight tables. This method was used to find a majority of the exons in selected known genes, and also predicted potential new exons which may be used in alternative splicing situations.     

Analysis of canonical and non-canonical splice sites in mammalian genomes

A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus approximately 600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.     

A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing.

We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.