Applications Turning Junk into Gold

The title of this subsection has been borrowed from that of an essay on the practical application of introns and inteins (Wickelgren 2003). Basically, the topic breaks down into the utility of homing endonucleases, group II intron RNPs, and inteins.

5.1 Site-Specific Group I Intron and Intein Endonucleases

The engineering of DNA often demands cleavage at rare sites and, to some extent, the highly site-specific endonucleases of group I introns and inteins fulfill the requirement. One need simply open the catalog of a molecular cloning company to identify those enzymes that cut the bacterial genome of -5000 kb seven times, or a yeast genome of -13,000 kb only once. They sound like restriction enzymes, bearing odd names like I-Crel and PI-PspI, with the prefixes I and PI, respectively, designating intron-encoded and protein intron-en-coded (Perler, this Vol.). The engineering of single sites of the historic a in-tron-endonuclease, I-Scel, into everything from yeast to mammals has facilitated not only genome sequencing projects, but also studies of DSB repair and non-homologous end joining (Dujon, this Vol.). However, the dream is not only for rare cleavage, but also for customized recognition sequence specificity. LAGLIDADG endonucleases are indeed starting to be engineered to alter their recognition-site specificity, through domain shuffling, and selection for new amino acid-DNA contacts, as described by Gimble later in this volume (Fig. 3A).

5.2 Gene Targeting by a Group II Intron RNP Complex

The novel DNA insertion mechanism of group II introns, via intron RNA-target DNA base pairing, has enabled the development of group II introns as site-specific gene targeting agents (Lambowitz et al., this Vol.). Through genetic manipulation, the intron RNA sequences that base pair with DNA, the so-called exon binding sequences, are changed (Fig. 3B). Thereby the group II Ll.LtrB intron has been re-targeted to specific genes in several bacteria. Additionally, this intron has been directed to HIV provirus and a plasmid-borne HIV coreceptor, and remained functional in transfection assays. This group II intron system is poised for different kinds of targeted gene manipulation (Fig. 3B), including gene disruption by integration into the antisense of an expressed gene, and conditional disruption via insertion into the sense strand and regulation of splicing. Such approaches will greatly facilitate functional

I-Crel homodimer monomer

1-0 mol heterodimer

HOMING TARGET

Gene disruption

NEW TARGET

publications application publications reviews

Fig. 3. Practical application of introns and inteins. a Domain manipulation of group

I intron endonucleases. The splitting of monomers into heterodimers and the generation of hybrid nonomers pave the way for increasing the substrate repetoire of LAGLI-DADG homing endonucleases (Gimble, this Vol.). b Group

II introns as gene-targeting agents. The wild-type intron lariat targeting its natural homing site through its exon binding sequence (EBS; both in black) is shown on the left. Mutation of the EBS to target new sites is reflected on the right by a color change of the EBS that matches that of the new site (blue or green). Gene disruption, which can be either absolute (-) or conditional (+), is shown at the top, and delivery of a foreign cargo gene, with maintenance of gene function (+), is shown at the bottom (Lambowitz et al., this Vol.). c Burgeoning intein technology since the discovery of inteins in 1990 (Perler, this Vol.).

genomics. Gene therapy, involving delivery of a foreign sequence, is a major goal, pending delivery to mammalian cells, transport through the nuclear envelope, and integration into the desired chromosomal sites.

5.3 "Inteins ... Nature's Gift to the Protein Chemist"

The metaphor in this subheading derives from a recent comment made by in-tein researcher Tom Muir (2004, pers. comm.). In the short time span since their discovery barely 15 years ago, intein activity has been harnessed and controlled to build an entirely new area of biotechnology (Fig. 3C), which is predicted to explode in the coming years. Intein technology has already facilitated protein purification, as well as providing tools for the study of proteins both in vitro and in vivo.

Switches that control splicing range from temperature and pH shifts (Wood et al., this Vol.) to the addition of small molecules, such as the reducing agent dithiothreitol (Chong and Xu, this Vol.), rapamycin and 4-hydroxytamoxifen (reviewed by Ozawa and Umezawa, this Vol.). In all cases, the intein was modified in order to impart controllability. For facilitation of such intein engineering, and for the monitoring of intein activity in vitro and in vivo, a battery of different reporter systems have been developed (Chong and Xu, this Vol.; Wood and Skretas, this Vol.).

By using the basic chemistry of the intein, mutant intein-protein fusions can be chemically ligated to a protein or peptide with an N-terminal cysteine residue. This process has been variously termed expressed protein ligation (EPL) or intein-mediated protein ligation (IPL) (reviewed by Perler later in this volume). This EPL/IPL technology has already had enormous application, as for example in protein stabilization and potentiation by cyclization (Tavas-soli et al., this Vol.), segmental labeling for NMR, or tagging with different reporters (Muir 2003). Equally impressive are in vivo intein-based technologies, many of them based on split inteins, and many using fluorescence or light as the reporter (Ozawa and Umezawa, this Vol.). These include monitoring of intracellular protein-protein interactions; identification of proteins specifically imported into mitochondria, endoplasmic reticulum, or nucleus; and even generation of safer transgenic plants.

This staggering array of technology that has developed recently is underscored by the increasing numbers of papers dedicated to the subject (Fig. 3C). Looking into the crystal ball, we can envision the proliferation of intein-based biosensors (Chong and Xu, this Vol.), proteomics utilizing peptide arrays (Ta-vassoli et al., this Vol.; Wood et al., this Vol.), and even functional proteomics in whole animals (Ozawa and Umezawa, this Vol.). Nature's gift, indeed!

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