In vivo expression of the nucleolar group I intron-encoded I-DirI homing endonuclease involves the removal of a spliceosomal intron

Abstract

The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3Ј end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5Ј end, as well as truncation and polyadenylation downstream of the ORF 3Ј end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA.

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

“…GIR1-dependence of endonuclease expression from the intact intron+ A: Schematic drawing depicting the PCR-based endonuclease assay+ A PCR product is incubated with extract+ If endonuclease is present, it cleaves the substrate, producing the unique five-base 39 overhangs specific to the Naegleria NaSSU1 endonuclease+ A double-stranded oligonucleotide (indicated by dark bars) with overhangs complementary to one of the products is added and can be ligated to a cleavage product+ The resulting DNA is amplified by PCR+ Small arrows represent primers+ B: Results of PCR based I-NjaI assay+ Lanes 1 and 11: 100-bp ladder; lanes 2-4: 50, 25, or 5 mg, respectively, of total protein from galactose-induced cells carrying the wild-type plasmid pYGal-NjaSSU; lanes 5-7 and 8-10: 50, 25, or 5 mg, respectively, of total protein from independently transformed, galactose-induced cells carrying the GIR1-crippled plasmid pYGal-G1 Ϫ Nja+ The specific product for the hybrid is indicated by the arrow+ An ;750-bp band in each lane (indicated by a circle on the right) is a PCR artifact+ the intron-homing endonuclease+ To address this hypothesis, we have examined intron-RNA processing in N. gruberi, a host organism for NaSSU1+ The data presented here show that in vivo cleavage also occurs just upstream of the ORF, at both the IPS1 and the IPS2 sites identified in vitro+ As primer extension showed only the 59 end corresponding to IPS2, the sequential self-cleavages reported by Einvik and colleagues (1998b) in vitro must occur very efficiently in Naegleria+ A result similar to ours was obtained in the analysis of DiSSU1 in its host organism D. iridis (Vader et al+, 1999)+…”

“…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...…”

“…We have shown previously that the large intron inserted in the gene for the small subunit ribosomal RNA of certain Naegleria species (e+g+, N. gruberi strain NB-1) consists of two group I ribozymes (Einvik et al+, 1997)+ Specifically, the larger self-splicing ribozyme (NaGIR2) is formed by interactions between 59 and 39 segments of the intron+ This arrangement places the small ribozyme NaGIR1 and the endonuclease ORF in the P6 loop of NaGIR2+ The translation-initiation codon for the N. gruberi endonuclease (I-Ngr I) is over 400 nt downstream from the 59 SS with six upstream AUG codons preceding the initiation codon+ In vitro transcripts of the intron RNA undergo self-cleavage mediated by GIR1, thereby eliminating most of the RNA segment upstream of the start codon for I-Ngr I+ Two closely spaced hydrolysis sites (internal processing sites IPS1 and IPS2) have been identified a few nucleotides upstream of the start codon (Einvik et al+, 1997(Einvik et al+, , 1998b)+ We reasoned that if this processing occurs in vivo, self-cleavage could influence expression of the homing endonuclease+ To examine whether NaGIR1 catalyzes internal processing of NaSSU1 RNA in its natural host organism, we performed Northern blot analysis on total RNA from NB-1 (intron ϩ) and EG B (intron Ϫ) amoebae+ An exon probe as well as simple observation of ethidium-bromide-stained RNA confirmed that NaSSU1 is efficiently spliced from the preribosomal RNA to produce an abundance of NB-1 SSU rRNA equal in size to the 2,019-nt EG B SSU rRNA (data not shown)+ Probes covering different portions of the 1+3-kb intron (shown schematically in Fig+ 1A, top) yielded distinct patterns of bands on the blots (Fig+ 1B)+ With an intron probe specific to the ORF, an abundant small RNA of ;900 nt was seen, as well as a slightly smaller and less abundant species of ;790 nt, in addition to large RNAs corresponding to the primary transcript and excised intron intermediates+ With an intron probe specific to sequences upstream of the internal processing sites IPS1/IPS2, an RNA species of ;300 nt was seen, as well as a small amount of an RNA of ;400 nt, in addition to RNAs corresponding to the primary transcript and excised intron intermediates+ The assignment of primary structure to these several bands is shown schematically in the margins of Figure 1+ These same patterns of bands were also observed in Northern blots when RNA was extracted from N. gruberi NB-1 that had been induced to differentiate into flagellates by shaking washed amoebae in a nutrient-free suspension for 80 min (Fulton, 1970; data not shown)+ The small RNAs visualized by the upstream probe imply that the intron RNA was further processed in the region proximal to IPS1/IPS2, and primer-extension analysis identified a 59 end at nt 125 of the intron (data not shown)+ Accumulation of the 790-nt RNA seen with the probe against the ORF suggests that some of the liberated ORF-containing RNA fragments undergo an additional processing event+ The Northern blot data combined with an S1 mapping assay (data not shown) are consistent with processing approximately 40 nt downstream of the ORF+ Interestingly, the 790-nt Naegleria RNA appears analogous to the ;850-nt polysomal I-Dir I mRNA from the related twin-ribozyme intron DiSSU1 (Vader et al+, 1999)+ The efficient formation of full-length intron circles has been observed in vitro with wild-type versions of the NaSSU1 intron, but not with deletion mutants lacking GIR1 and ORF (Einvik et al+, 1997)+ To test if such RNA circles are also formed in vivo, we employed an RT-PCR approach like that designed previously …”

“…The exact mechanism by which proteins encoded by group I introns embedded in eukaryotic rRNA genes are expressed has not been elucidated+ In the case of the twin-ribozyme intron DiSSU1 in its natural host D. iridis, maturation of the endonuclease mRNA was found to include internal processing by the GIR1 ribozyme, as well as removal of a spliceosomal intron and polyadenylation (Vader et al+, 1999)+ Perhaps the beststudied intron of this type to date is PpLSU3, artificially inserted into all the rDNA repeats of yeast (Lin & Vogt, 1998;2000)+ In this system, it was inferred that the intact excised intron RNA, and not an RNA species that is processed further, is the source of the message for the I-PpoI endonuclease, whose coding region starts 14 nt downstream of the 59 splice site (SS)+ By contrast, in the twin-ribozyme group I introns, the proteinencoding region is not positioned close to the 59 SS, and numerous AUG codons occur between the 59 end of the RNA and the start of the ORF+ According to the scanning mechanism of eukaryotic translation initiation, upstream AUGs should greatly attenuate or eliminate expression of the ORF (Sachs et al+, 1997)+ Certain viral and a few nuclear messengers have evolved complex elements called internal ribosome entry sites to recruit the ribosome (Belsham & Sonenberg, 1996;Sachs et al+, 1997), and hence a priori it is conceivable that nuclear group I introns use such a mechanism for translation as well+…”

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

“…Here we suggest that the K-motif in P9.2 represents a key regulatory element between the reaction pathways, and that a protein factor may exists in the Didymium nucleus that participates in the regulation. The observation that splicing appears much more efficient than hydrolysis in vivo compared to in vitro [8,[12][13][14] is consistent with this proposal.…”