Characterization of the self-splicing products of a mobile intron from the nuclear rDNA of Physarum polycephalum.
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.
Ruoff
B
B
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
Johansen
S
S
Vogt
V M
VM
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000378:metabolism; D004275:DNA, Ribosomal / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D007438:Introns; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016839:Physarum polycephalum / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D015183:Restriction Mapping
D004271:DNA, Fungal; D004275:DNA, Ribosomal