We have learned a lot about inteins, but there is much more to discover. Mechanisms for splicing of non-canonical inteins still need to be refined. How inteins control the order of reactions in the splicing pathway is still to be determined. The active site of inteins needs to be further analyzed to understand the mechanism of splicing at a more detailed level. Structures of intermediates and active precursors are needed. Many basic questions about evolution of inteins are still unanswered, including the role of inteins in early evolution, how inteins became associated with homing endonucleases, how intein insertion sites and homing endonuclease recognition sites merged, or how inteins spread across phylogenetic domains. Is the absence of inteins in multicellular organisms due to barriers preventing lateral transmission to germ cells? Although inteins have not been found in higher organisms, hedgehog auto-processing domains are present. The diversity of intein applications is growing exponentially. The future is only limited by our imagination.
Acknowledgements. I thank Marlene Belfort for initiating the intein nomenclature project that led to InBase as a resource for those interested in inteins and the many protein splicers that continue to contribute to InBase. I pay homage to the creativity displayed by researchers developing intein applications whose contributions are too numerous to include herein, especially Tom Muir. I apologize to all those whom I have not mentioned due to space limitations. I thank Ming Xu who came to my laboratory as a postdoc with an enthusiasm for determining the protein-splicing mechanism and Maurice Southworth who has been part of my group since the time before inteins and who has enjoyed playing with them as much as I have. I thank Donald Comb for supporting my research.
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