Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme

NaSSU1 is a complex nuclear group I intron found in several species of Naeg leria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an op en reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites abo ut 400 nt downstream of the 5' end of the intron, proximal to the endonucle ase ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in g ene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant y east strain was selected with a mutation in all the rRNA genes, rendering t he rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuc lease, which is otherwise lethal, could be expressed readily in these cells . Endonuclease activity also could be detected in extracts of yeast harbori ng plasmids in which the endonuclease ORF was embedded in its native contex t in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constru cted to prevent self-cleavage of the RNA failed to express endonuclease act ivity. These results support the hypothesis that the NaGIR1-catalyzed self- cleavage of the intron RNA is a key event in expression of the endonuclease .