I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Abstract

PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023-1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as the Tetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.Group I introns are a class of RNA elements that share a secondary structure which allows the intron to undergo selfsplicing from the primary transcript (5). While most group I introns are located in the genes of mitochondria and chloroplasts of lower eucaryotes, some are found in nuclear genes. Interestingly, nuclear group I introns reside only in rDNA, the gene encoding rRNA, and when present they occupy all of the ca. 200 rDNA copies typical of eucaryotic organisms. Some group I introns are mobile genetic elements. They encode a site-specific endonuclease that recognizes and cleaves a DNA sequence at or near the intron insertion site of the intronlacking allele. The double-strand break is then repaired by replication of the intron into the intron-lacking allele, thus converting all intron-lacking alleles into intron-containing alleles. This process is termed intron homing due to its high specificity (3).Among the ca. 150 nuclear group I introns reported so far, only three have been shown or have been inferred to be mobile: DiSSU1 from the slime mold Didymium iridis (6, 21, 22), NaSSU1 from the protist Naegleria andersoni and other Naegleria species (8), and PpLSU3 from the slime mold Physarum polycephalum. Originally found in the large-subunit rDNA gene of the Carolina strain (29), PpLSU3 contains the open reading frame (ORF) for the homing endonuclease I-PpoI (for nomenclature of intron-encoded endonucleases, see reference 7) in its 5Ј half and the ribozyme part in its 3Ј half. The sequence of the ribozyme part of PpLSU3 is 70% identical to the Tetrahymena thermophila intron TtLSU1, which is inserted at the same location as PpLSU3, suggesting a common evolutionary origin. Previous work has shown that PpLSU3 RNA not only undergoes self-splicing but also cleaves itself at an internal processing site (IPS), thus separating the I-PpoI ORF and the ribozyme (38). I-PpoI recognizes a 13-to 15-bp DNA sequence in a portion of the large-subunit rRNA gene that is 100% identical in all eucaryotes (9, 52). When a plasmid-borne PpLSU3 is transformed into Saccharomyces cerevisiae, most cells die upon the induction of I-PpoI expression, because I-PpoI makes double-strand breaks in the ca. 120 rDNA repeats on chromosome...

Pentamidine Inhibition of Group I Intron Splicing in Candida albicans Correlates with Growth Inhibition

“…This effect may be due to regulatory processes that inhibit all later steps in the maturation pathway when an early step (intron removal) is blocked. This is consistent with the finding that insertion of an unspliceable mutant intron derived from Pp.LSU3 into the rRNA gene of S. cerevisiae results in accumulation of two ITS2-containing 27S rRNA precursor species (21).…”

Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast

“…The 413 nucleotides (nt) intron is inserted after helix 69 in the large subunit rRNA (position 1925, E. coli numbering), a conserved region of the large subunit rRNA that directly contacts the small ribosomal subunit (Cate et al 1999). Lin and Vogt (1998) found that the Tetrahymena intron spliced efficiently in the yeast Saccharomyces cerevisiae, when transposed into the 25S rRNA gene at the position homologous to its natural splice junction in Tetrahymena. Cells remained viable when the intron was integrated into all of the chromosomal rDNA repeats, demonstrating that the spliced 25S rRNA was functional.…”

“…cerevisiae strain INVSc2/TtLSU1 (MATa his3-D200 ura3-167 25S::TtLSU1) has the Tetrahymena group I intron integrated into every copy of the 25S rDNA on Chromosome XII (Lin and Vogt 1998), and was the gift of V. Vogt. For splicing assays, plasmids expressing the intron were transformed into strain BY4733 (MATa his3D200 leu2D0 met15D0 trp1D63 ura3D0).…”

“…These sequences were amplified by PCR and subcloned into the BamHI and XhoI sites of p425-GAL1. Plasmid pSJ015 is the same as pSW015 except the flanking rRNA exon sequence is derived from the yeast 25S rRNA, amplified from the rDNA of INVSC-TtLSU1 (Lin and Vogt 1998). Point mutations were introduced into pSJ015 by exchanging the SphI-NheI intron fragment with variants of pSW012 carrying the desired mutation (Pan and Woodson 1998).…”

“…In Tetrahymena thermophila, the intron is located in the large subunit (26S) rRNA and self-splices early during pre-rRNA processing (Cech and Rio 1979;Din et al 1979), with an apparent t 1/2 z2 sec (Brehm and Cech 1983). The Tetrahymena intron was similarly reactive when inserted into the Escherichia coli 23S (Zhang et al 1995) or yeast 25S rRNA (Lin and Vogt 1998). In contrast, splicing is 20-200 times slower in vitro (t 1/2 = 0.7-7 min) because up to 95% of the pre-rRNA misfolds when transcribed in vitro (Bass and Cech 1984;Emerick and Woodson 1993).…”