Protein splicing
The splicing junction of the precursor protein is mainly a cysteine or a serine, which are amino acids containing a nucleophilic side chain.
[3] In 1990 Hirata et al.[4] demonstrated that the extra sequence in the yeast gene was transcribed into mRNA and removed itself from the host protein only after translation.
[7][8] As of December 2019, the UniProtKB database contains 188 entries manually annotated as inteins, ranging from just tens of amino acid residues to thousands.
They were later found in fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins as well.
Not surprisingly, most intein in eubacteria and archaea are found to be inserted into nucleic acid metabolic protein, like fungi.
For example, pre-mRNA processing factor 8 (Prp8) protein, instrumental in the spliceosome, has seven different intein insertion sites across eukaryotic species.
[14] Intein-containing proteins found in archaea include RadA (RecA homolog), RFC, PolB, RNR.
A transesterification occurs when the side chain of the first residue of the C-extein attacks the newly formed (thio)ester to free the N-terminal end of the intein.
This forms a branched intermediate in which the N-extein and C-extein are attached, albeit not through a peptide bond.
An O-N or S-N shift produces a peptide bond and the functional, ligated protein.
[17] Class 3 inteins have no nucleophilic first side chain, only an alanine, yet they have an internal noncontiguous "WCT" motif.
Several studies have demonstrated the modular nature of inteins by adding or removing HEG domains and determining the activity of the new construct.
For example, in cyanobacteria, DnaE, the catalytic subunit α of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c.
It has been suggested that inteins could prove useful for achieving allotopic expression of certain highly hydrophobic proteins normally encoded by the mitochondrial genome, for example in gene therapy.
This problem can be avoided by fusing an affinity tag to self-cleavable inteins in a controlled environment.
The modified intein undergoes a self-cleavage reaction at its N-terminal peptide linkage with 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME), or cystine at low temperatures over a broad pH range.
This novel technique eliminates the need for a proteolysis step, and modified Sce VMA stays in column attached to chitin through CBD.
[12] Intein splicing is found exclusively in unicellular organisms, with a particularly high abundance in pathogenic microorganisms.
For example, Gaëlle Huet et al. demonstrated that in Mycobacterium tuberculosis, unspliced SufB prevents the formation of the SufBCD complex, a component of the SUF machinery.
Current research on intein splicing inhibitors has focused on developing antimycobacterials (M. tb.
[12] In 2021, Li et al. showed that small molecule inhibitors of Prp8 intein splicing were selective and effective at slowing the growth of C. neoformans and C. gattii, providing exciting evidence for the antimicrobial potential of intein splicing inhibitors.
