We have characterized the splicing products formed in vitro from RNA derived from the mobile group I intron in the nuclear rDNA of Physarum polycephalum, Pp LSU 3. This intron is a close relative of the well known Tetrahymena intron Tt LSU 1, being inserted at exactly the same position in the rDNA and sharing about 90% sequence identity with Tt LSU 1 in the conserved elements characteristic of the catalytic core of all group I introns. However, Pp LSU 3 differs from Tt LSU 1 in that it encodes a site-specific endonuclease, which mediates the homing of the intron to unoccupied target sites. The endonuclease, I-Ppo, would appear to be a unique example of a protein encoded by an RNA polymerase I transcript. To gain clues to the splicing products formed in vivo, and to the nature of the messenger RNA for I-Ppo, we subjected Pp LSU 3 RNA to standard self-splicing conditions in vitro, and then analyzed the products by size, by northern blotting, and by primer extension. The results show two novel features. First, in addition to the expected 5' splice site, there is an alternative 5' splice site in the upstream exon, just preceding the first codon of the I-Ppo open reading frame. Second, at the position corresponding to the major circularization site in Tt LSU 1 there is an internal processing site, leading to the efficient separation of two halves of the excised intron, the 5' half encoding I-Ppo and 3' half containing the ribozyme. Surprisingly, this cleavage appears not to be due to circularization followed by hydrolytic opening of the circle, but rather to G addition. The formation of these products in vitro suggests how the messenger RNA for the I-Ppo endonuclease may be generated in vivo.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.