Group I twintrons: Genetic elements in myxomycete and schizopyrenid amoeboflagellate ribosomal DNAs

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

“…Under conditions used to force homing of PpLSU3 into yeast rDNA (Muscarella & Vogt, 1993;Lin & Vogt, 1998), NaSSU1 failed to home successfully in yeast+ PCR analysis did not detect any intact introns in rDNA+ However, this analysis did provide evidence of rare intron-exon junction fragments, suggesting that homing at least had been initiated+ Either gene conversion then failed to proceed to completion, or after gene conversion was completed at least in some rDNA copies, the introns were deleted or partially deleted because of deleterious effects on cell growth+ The latter possibility is more likely: we showed that when NaSSU1 was artificially built into a plasmid-born rDNA, preribosomal RNA expression from the plasmid was not able to rescue cell growth in the absence of RNA polymerase I transcription of the rDNA copies on chromosome XII (data not shown)+ It is possible that this failure is because of lack of splicing, or inefficient or incorrect splicing+ Alternatively, the intron might affect some other aspect of ribosomal RNA maturation+ The latter possibility is suggested by the observation that the Tetrahymena intron inserted into the ribosomal RNA genes of Schizosaccharomyces pombe prevented proper maturation of the 5+8S species, despite the absence of obvious splicing defects (Good et al+, 1994)+ Presumably group I introns in nuclear rDNA have coevolved with rDNA to maximize splicing efficiency and to minimize disruption of other processes in their host organisms+ It seems possible that minor sequence changes might allow them to adapt to new species, but this notion has not been tested experimentally+ To study NaSSU1 splicing, processing, and endonuclease expression in yeast, we developed a strategy that circumvented the lack of homing in this system+ First, the endonuclease was expressed by itself from a plasmid, and among surviving colonies, a mutant yeast strain was identified that tolerates endonuclease activity+ This strain had acquired a single mutation in the endonuclease target site in all the rDNA copies, analogous to the mutations described previously that confer resistance to the I-PpoI endonuclease in yeast (Muscarella & Vogt, 1993;Lin & Vogt, 1998)+ Into this strain a plasmid was introduced that carries NaSSU1 with ;0+3 kb flanking rDNA 59 exon and 39 exon sequences, under control of the GAL1 promoter+ Induction by galactose in this system led to the synthesis of an rRNA from which the intron spliced and further processed itself, in a manner similar to that seen in vitro and also in Naegleria+ Furthermore, intron expression led to endonuclease activity, which could be detected in crude extracts either by a straightforward DNAcleavage assay, or by a more sensitive PCR-based assay for the characteristic 5 nt sticky end produced by cleavage+ An inactivating mutation in the NaGIR1 ribozyme abrogated endonuclease activity+ This result thus supports the hypothesis that the function of NaGIR1 is to generate the mRNA for the Naegleria endonuclease+ NaSSU1 and DiSSU1 are the only known examples of twin-ribozyme group I introns+ Despite differences in organization (Einvik et al+, 1998a), comparison of processing reveals a number of shared features+ First, in both the DiSSU1 and NaSSU1 systems, a GIR1-mediated cleavage is observed in vivo downstream of the ORF+ Second, both DiSSU1 RNA (Vader et al+, 1999) and NaSSU1 RNA form full-length circles in their host organisms+ Circularization, like 39 SS hydrolysis, is usually thought to be a side reaction of...…”

“…Group I introns are phylogenetically widespread autocatalytic RNA elements sharing a common secondary and tertiary structure (Cech et al+, 1994)+ Many group I introns are capable of self-splicing in vitro through a series of transesterification reactions (Cech, 1990), although proteins may aid splicing in vivo (Lambowitz & Perlman, 1990;Shaw & Lewin, 1997)+ About one third of group I introns harbor open reading frames (ORFs) coding for either structural proteins, maturases, or DNA endonucleases (Johansen et al+, 1997a)+ The DNA endonucleases, which are the most common intron-encoded proteins, are usually involved in intron mobility at the DNA level (reviewed in Belfort & Roberts, 1997)+ The endonuclease creates a doublestrand break at the intron-lacking allele, which is subsequently repaired in a gene-conversion event, using the intron-containing allele as a template+ The resulting unidirectional transfer of intron sequences is termed intron homing because of its high specificity at a homologous site+ Only 5% of the ;300 reported nuclear rDNA group I introns contain ORFs, and among these are the optional PpLSU3 and DiSSU1 of the myxomycetes Physarum polycephalum and Didymium iridis, respectively (Muscarella & Vogt, 1989;Johansen & Vogt, 1994), and NaSSU1 in a few species of the free-living amoebaflagellate Naegleria (De Jonckheere, 1994)+ These introns interrupt rRNA genes on extrachromosomal rDNA molecules (see Einvik et al+, 1998a), and encode the functional site-specific His-Cys box homing endonucleases named I-PpoI, I-Dir I, and I-NjaI, respectively (Muscarella et al+, 1990;Johansen et al+, 1997b;Elde et al+, 1999)+ I-PpoI and I-Dir I are involved in homing in their natural hosts after mating (Muscarella & Vogt, 1989;Johansen et al+, 1997b)+ NaSSU1 and DiSSU1 constitute a unique class of group I introns referred to as twin-ribozyme group I introns (Decatur et al+, 1995;Einvik et al+, 1997Einvik et al+, , 1998a)+ In these introns, the endonuclease ORF is found downstream of a small group I self-cleaving ribozyme (GIR1) that catalyzes hydrolytic cleavage of the RNA just upstream of the endonuclease ORF (see Fig+ 1)+ These two elements are both embedded in a loop of a more regular group I self-splicing ribozyme (GIR2)+ Strikingly, some species of Naegleria contain a shorter version of NaSSU1 that lacks both the ORF and GIR1 (De Jonckheere & Brown, 1994;Einvik et al+, 1997), suggesting that these two elements form a functional genetic unit, acquired or lost at the same time in evolution+ Despite their small si...…”

The ability to form full-length intron RNA circles is a general property of nuclear group I introns

“…During in vitro processing of Dir.S956-1, the corresponding intermediates and products accumulate as expected for the circularization pathway (Johansen and Vogt 1994;Decatur et al 1995;Einvik et al 1998a). In vivo, the product of the circularization pathway is easily detectable by Northern blotting analysis (Vader et al 1999(Vader et al , 2002.…”

“…These introns contain a canonical group I splicing ribozyme (GIR2) harboring a selfcleaving group I-like ribozyme (GIR1) and a homing endonuclease gene (HEG) inserted into a peripheral loop (for review, see Einvik et al 1998a;Johansen et al 2002). Our studies of the intron in Didymium iridis (Dir.S956-1) have revealed that the structural complexity is paralleled by a complex biology.…”

Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing

“…Hydrolytic cleavage at the 3¢-SS is initiated when the last intron nucleotide (TG) binds to the GBS prior to exoG. Splicing and hydrolysis are competing reactions leading to ligated exons and full-length intron circles, respectively [7].We have identified and examined an unusual category of self-splicing group I introns with a complex structural organization and function [8][9][10][11]. These twin-ribozyme introns consist of two distinct ribozymes (GIR1 and GIR2) and a homing endonuclease gene (HEG).…”