In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.     

Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.     

RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration

Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.     

Protein splicing in the absence of an intein penultimate histidine

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.     

Protein splicing: its discovery and structural insight into novel chemical mechanisms

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure-directed chemistry, a series of crystal structural studies has been carried out with the use of splicing-inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N-terminal junction of the introduced extein polypeptide forms an intermediate containing a five-membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N- and C-extein segments. This article summarizes biochemical and structural studies on a self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation.     

Requirement of protein factors and ATP for the disassembly of the spliceosome after mRNA splicing reaction.

Pre-mRNA splicing reaction occurs in a large ribonucleoprotein complex called the spliceosome. After the splicing reaction, the spliceosome is disassembled to release the splicing products including spliced mRNA. Here we show that protein factors in a HeLa nuclear extract or a DEAE-cellulose fraction as well as ATP are required to release the splicing products form the spliceosome in which the splicing reaction has already completed.     

Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction

We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction.     

Role of primary structure and disulfide bond formation in beta-lactamase secretion

Plasmid pBR322-encoded beta-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfate-polyacrylamide gels than the fully reduced form. A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond. Formation of the disulfide bond in vivo, however, occurred concomitant with processing. In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur. This result is consistent with post-translational translocation of the precursor. Synthesis of a fragment of beta-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla. Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion.     

Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.

An SP6/mouse insulin RNA precursor containing two exons and one intron can be spliced in a partially purified nuclear extract isolated from MOPC-315 mouse myeloma cells. We have detected the putative RNA splicing intermediate (intron-3'exon) in a lariat form, the excised intron in a lariat form, and the mRNA spliced product. The in vitro splicing reaction of gel-purified RNA precursors requires ATP and Mg2+ and was accompanied by the formation of a 60-40S ribonucleoprotein complex. The formation of the 60S complex requires ATP. At least two Sm snRNPs containing U1 and U2 RNAs are components of the 60-40S complex. The assemble of those snRNPs occurs early during the splicing reaction and it requires ATP and intron containing pre-mRNAs.     

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.     

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.     

Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence.

The M. tuberculosis recA locus comprises an 85 kd open reading frame but produced 38 kd RecA and 47 kd products in E. coli. No RNA processing was detected; rather, an 85 kd precursor protein was spliced, releasing a 47 kd spacer protein, and joining its terminal fragments to form mature RecA protein. "Spacer" protein was also produced in M. tuberculosis and from a hybrid spacer-LacZ alpha fusion molecule. Mutagenesis at codon wobble positions at one splice junction showed that protein rather than nucleotide sequence determined splicing activity. Other mutants defined additional regions needed for splicing and allowed processing to be followed. Splicing was essential for RecA activity in E. coli. The possibility that splicing is a manifestation of a novel class of genetic element is discussed.     

A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction.

tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.