Characterization of the self-splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes Peik Haugen 1 , Johan F. De Jonckheere 2 and Steinar Johansen 1 1 RNA Research group, Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway; 2 Protozoology Laboratory, Scientific Institute Public Health – Louis Pasteur, Brussels, Belgium The two group I introns Nae.L1926 and Nmo.L2563, found at two different sites in nuclear LSU rRNA genes of Naegleria amoebo-flagellates, have been characterized in vitro. Their structural organization is related to that of the mobile Physarum intron Ppo.L1925 (PpLSU3) with ORFs extending the L1-loop of a typical group IC1 ribozyme. Nae.L1926, Nmo.L2563 and Ppo.L1925 RNAs all s elf- splice in vitro, generating ligated exons and full-length intron circles as well as internal processed excised intron RNAs. Formation of full-length intron cir cles is found to be a general feature in RNA processing of ORF-containing nuclear group I introns. Both Naegleria LSU rDNA introns contain a conserved polyadenylation signal at exactly the same position in the 3¢ end of the ORFs close to the internal processing sites, indicating an RNA polymerase II-like expression pathway of intron proteins in vivo. The intron proteins I-NaeIandI-NmoI encoded by N ae.L1926 and Nmo.L2563, respectively, correspond to His-Cys homing endonucleases of 148 and 175 amino acids. I-NaeIcontains an additional sequence motif homologous to the unusual DNA binding motif of three antiparallel b sheets found in the I -PpoI endonuclease, the product of t he Ppo.L1925 intron ORF. Keywords: group I ribozyme; mobile intron; ribosomal DNA; R NA processing. About 3% of the  850 nuclear group I i ntrons in the database contain large ORFs or ORF-like sequences inserted into peripheral loop regions of their corresponding group I ribozymes. Whereas ORFs encoding proteins with a possible structural r ole have been noted in the green alga Scenedesmus [1] and the fungus Protomyces [2], most nuclear group I intron ORFs correspond to endonucleases (Table 1). All the nuclear endonucleases contain a con- served histidine and cysteine rich motif [3,4] directly involved in zinc-binding and the active site of the enzymes [5,6]. The biological r ole of group I intron endonucleases appears to b e i n i ntron h oming a t the D NA l evel [7]. Homing is initiated by a double-strand break made by the endonuclease at an i ntron-less cognate site, proceeds by host-dependent gene conversion, and results in insertion of the group I intron by replication into the intron-less site. The endonucleases I-PpoIandI-DirI from nuclear group I introns Ppo.L1925 and Dir.S956-1 in the myxomycetes Physarum polycephalum and D idymium iridis have been reported to mediate intron homing in genetic crosses [8,9]. All known nuclear group I introns interrupt the highly expressed small ribosomal subunit (SSU) or large ribosomal subunit (LSU) rRNA genes, and have to be spliced out from the R NA polym erase I trans cribed p recursor r RNA. An intriguing question is thus how intron proteins encoded by nuclear group I i ntrons are expressed from an RNA polymerase I transcript. A protein encoding gene in a eukaryotic nucleus is in general t ranscribed by RNA polymerase II as premRNA. Here, pre-mRNA matu ration includes the addition of a methylated guanine to the 5¢ end (capping), the removal of spliceosomal introns, and poly- adenylation at the 3¢ end (reviewed in [10]). In vivo expres- sion analyses of the group I intron endonucleases I-PpoI, I-DirI, and I-NgrI indicate different s trategies and solutions [11–13]. Based on Ppo.L1925 trans-integration in yeast rDNA, I-PpoI mRNA was shown to be transcribed by RNA polymerase I and subsequently translated from the excised, but unprocessed, intron RNA [11]. Furthermore, the messenger appeared not to be polyadenylated [14], a nd sequences downstream the I- PpoI O RF RNA , preceding the group I ribozyme, were found to be important in both splicing a nd protein expression [15]. Expression of I-DirI and I-NgrI from twin-ribozyme introns [16] is dependent on novel group I-like ribozymes responsible for the formation of the 5¢end of their mRNAs [12,13], and examination of polysome a ssociated I- DirI m RNA s upports that matur- ation also includes the removal of a 51 nucleotide spliceo- somal intron and polyadenylation [12]. Many group I introns self-splice as naked RNA in vitro, catalyzed by intron-encoded group I ribozymes. The intron sequences are excised from precursor RNA by a two step trans-esterification reaction, w ith a subsequent ligation of flanking exon sequences [17]. Additional ribozyme-cata- lyzed RNA processing reactions, including intron circular- ization a nd internal processing, have b een characterized in Correspondence to S. Johansen, Department of Molecular Biotech- nology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway. Fax: + 47 77 64 53 50, Tel.: + 47 77 64 53 67, E-mail: steinarj@fagmed.uit.no Abbreviations: LSU, large ribosomal subunit; premRNA, precursor messenger RNA; rDNA, ribosomal DNA; rRNA, ribosomal RNA; SSU, small ribosomal subunit. (Received 26 October 2001, revised 7 January 2002, accepted 22 January 2002) Eur. J. Biochem. 269, 1641–1649 (2002) Ó FEBS 2002 some intron systems that include the ORF-containing group I introns (Table 2). In vivo analyses of I-PpoI, I-DirI and I-NgrI expression in their original hosts and/or in yeast indicate an essential role of ribozyme-mediated intron RNA processing [11–13,15]. Two Naegleria species, Naegleria sp. NG874 and N. morganensis, contain large nuclear group I introns within the LSU rDNA encoding the putative homing endonucleases I-NaeIandI-NmoI, respectively. In order to gain insight into cellular maturation and processing patterns of the I-NaeIandI-NmoImRNAs,weanalysed their corresponding intron RNAs for self-splicing and self- processing in vitro. MATERIALS AND METHODS Plasmid construction, DNA sequencing, and computer analyses Introns are named according to the new proposed nomen- clature o f group I introns in ribosomal DNA [18] that include information of intron insertion site in the SSU (S) or LSU (L) ribosomal DNA genes. The Nae.L1926, Nmo.L2563 and Ppo.L1925 introns were PCR amplified from the corresponding Naegleria sp. (NG874 isolate), N. morganensis (NG236 isolate) and P. polycephalum (Carolina isolate) LSU rDN A segments using the primer sets OP460 (5¢-AATTAATACGACTCACTATAGGTCC TGCACACCTTGT-3¢)/O P461 (5¢-CGCCAGACTAGAG TCA-3¢), OP454 (5¢-AATTAATACGACTCACTATAGG CGGATAAGGCCAAT-3)/OP451 (5¢-GCTCACGTTCC CTGT-3¢), and OP452 (5¢-AATTAATA CGACTCACT AT AGGAACTTACAAAGGCTA-3¢)/ OP442 (5¢-GCCTTTC GAACGTCA-3¢), respectively. The PCR products, which contain the introns, some flanking exon sequences, and a primer generated T7 promoter, were cloned into pU C18 using the SureClone Ligation kit (Amersham Pharmacia Table 1. Nuclear group I introns with His-Cys box motif. Intron a and host Intron size (bp) ORF size (aa) b ORF location c Acc. no. Nja.S516 (Naegleria jamiesoni) 1307 245 P6/sense U80250 Nan.S516 (Naegleria andersoni) 1309 245 P6/sense Z15417 Nit.S516 (Naegleria italica) 1319 245 P6/sense U80249 Ngr.S516 (Naegleria gruberi) 1316 245 P6/sense X78278 Ncl.S516 (Naegleria clarki) 1305 245 P6/sense X78281 Nca.S516 (Naegleria carteri) 1324 245 P6/sense Y10190 Nae.S516 (Naegleria sp.NG 872) 1318 244 P6/sense AJ001399 Pte.S516 (Porphyra tenera) 972 162 P2/sense AB013175 Bfu.S516 (Bangia fuscopurpurea) 996 Pseudo P2/sense AF342745 Asp.S516 (Acanthamoeba sp.KA/E4) 957 Pseudo P2/sense AF349045 Emy.S943 (Ericoid Mycelia) 1755 Pseudo P8/sense AF158831 Mte.S943 (Monoraphidium terrestre) 1611 277 P8/a-sense ref [47] Dir.S956-1 (Didymium iridis) 1436 244* P2/sense X71792 Dir.S956-2 (Didymium iridis) 1203 192* P8/a-sense ref [46] Nga.S1199 (Nectria galligena) 1423 Pseudo P9/a-sense Y16424 Emy.S1199 (Ericoid Mycelia) 1330 Pseudo P9/a-sense Y158838 Pte.S1506 (Porphyra tenera) 960 Pseudo P1/a-sense AB013175 Psp.1506 (Porphyra spiralis) 1056 Pseudo P1/a-sense L26177 Bat.1506 (Bangia atropurpurea) 1038 Pseudo P1/a-sense L36066 Cal.L1923 (Candida albicans) 962 Pseudo P2.1/a-sense AB049125 Ppo.L1925 (Physarum polycephalum) 944 163 P1/sense L03183 Nae.L1926 (Naegleria NG874) 867 148 P1/sense AJ311176 Nmo.L2563 (Naegleria morganensis) 940 175 P1/sense AJ311175 a Named according to [18]. b Putative endonuclease Pseudogenes (Pseudo) due to frame-shifts/truncations. Estimated protein size after the removal of a small spliceosomal intron from pre-mRNA (*). c Group I ribozyme paired element (Pn) interrupted by endonuclease-like ORFs. ORF encoded by the same strand (sense) or opposite strand (a-sense) to that encoding the intron ribozyme and pre-rRNA. Table 2. RNA processing of ORF-containing nuclear group I introns. Intron Ligated exons a Full-length circles b In vitro/ in vivo Internal processing sites c in vitro/ in vivo Reference Nja.S516 + +/NA +/NA [43] Nan.S516 + +/NA +/NA [43] Nit.S516 + NA/NA +/NA [43] Ngr.S516 + NA/+ +/+ [13,43] Dir.S956-1 + +/+ +/+ [12,44,48] Dir.S956-2 + +/+ –/NA [46] Psp.S1506 + +/NA –/NA [1] Ppo.L1925 + +/NA +/+ [11,15,20], this work Nae.L1926 + +/NA +/NA This work Nmo.L2563 + +/NA +/NA This work a Confirmed (+) ligated exons (LE) by experimental approaches. b Confirmed (+) intron full-length circles (FLC) by experimental approaches. c Present (+) or absence (–) of ribozyme-cata- lyzed internal processing sites (IPS). Introns not analyzed (NA). 1642 P. Haugen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Biotech), yielding pT7Nae.L1926, pT7Nmo.L2563, and pT7Ppo.L1925, respectively. The inserts were sequenced using the Thermo Sequenase sequencing kit (Amersham Pharmacia Biotech) and [a- 33 P]ddNTPs (GATC; 450 lCiÆmL )1 ). The Nmo.L2563 i ntron w as found to be identical to the previously reported sequence [19], except for the addition of 21 nucleotides at the 3¢ end of the intron. The Nae.L1926 and Nmo.L2563 introns have been assigned the EMBL/GenBank Data library accession numbers AJ311176 and AJ311175, respectively. Computer analyses of nucleic-acid and amino-acid sequences were performed using the GCG software package programs from the Genetic Computer Group (Version 10; Madison, WI, USA), PHDSEC (Version 1.96; EMBL-Heidelberg, Germany), and PSIPRED (Version 2.0; Protein Bioinformatics Group, Brunel, UK). In vitro transcription and splicing The intron RNA was transcribed in vitro by T7 RNA polymerase from the linearized te mplates pT7Nae.L1926 (BamHI), pT7Nmo.L2563 (HindIII), and pT7Ppo.L1925 (HindIII). The RNA was uniformly labelled u sing [a- 35 S]CTP (10 lCiÆlL )1 ; Amersham Pharmacia Biotech), and subjected to self-splicing conditions (40 m M Tris pH 7.5, 0.2 M KCl, 2 m M spermidine, 5 m M dithiothreithol, 10 m M MgCl 2 and 0.2 m M GTP) at 50 °C for 0–30 min, all essentially as described previously [1]. Self-spliced RNA was subjected to electrophoresis in a 5% polyacrylamide, 8 M urea gel, and visualized by autoradiography. RNA circle and exon junction determination Ligated exon and circular i ntron RNAs were isolated from polyacrylamide gels and incubated in 400 lLelutionbuffer (0.3 M NH 4 Ac, 0.1 % SDS, 10 m M Tris pH 8 and 2.5 m M EDTA pH 8) on a rotating wheel at 4 °C over night. The RNA was purified using a 0.45-l M filter (Millipore), and ethanol precipitated. PAGE-purified RNA was subse- quently subjected to reverse transcription u sing the First Strand cDNA Synthesis kit (Amersham Pharmacia Biotech) and a downstream primer. Products were amplified by adding an upstream p rimer, then cloned into pUC18, and finally several clones of each product were DNA sequenced. Nae.L1926 intron RNA was analysed for ligated exon, full-length circle, and )15 circle using t he primer sets OP460 /OP461, OP460/OP463 (5 ¢-TAGAGCGGTAC TATA-3¢), and OP460/OP463, respectively. Nmo.L2563 intron RNA was analysed for ligated exon, full-length circle, and )551 circle using the primer sets OP450 (5¢-GCG GATAAGGCCAAT-3¢)/OP451, OP456 ( 5¢-GAGGCTAA ATCTCTTA-3¢)/OP494 (5¢-AGCTTTACTACACCT-3¢), and OP456/OP558 (5¢-CCCTACCTTACAGAT-3¢), res- pectively. Finally, the Ppo.L1925 f ull-length intron RNA circle was analysed by using the primer set OP444 (5¢-GGGTG C AGTTCACAGACT-3 ¢)/OP443 ( 5¢-ATGG TACATGGT GCGTTA-3¢). Mapping of internal processing sites The 5¢ ends of the internal p rocessing sites w ere mapped by primer extension as described previously [20,21]. The linearized plasmids pT7Nae.L1926 and pT7Nmo.L2563 were in vitro transcribed and submitted to self-splicing conditions for 60 min. The transcribed R NA was subse- quently p urified i n s everal steps including pheno l/chloro- form extraction, RQ1 DNase (Promega) digestion for 20 min at 37 °C followed by enzyme inactivation for 10 min at 70 °C, and finally separation in a MicroSpin S-400 HR column (Amersham Pharmacia Biotech). Purified Nae.L1926 and Nmo.L2563 intron RNAs were annealed to the oligo primers OP463 a nd OP558. The reverse transcrip- tion reactions were performed using the SuperScript II (Gibco BRL) enzyme with 10 lCi [a- 35 S]dCTP (Amersham Pharmacia Biotech) as the label. DNA sequencing ladders were prepared from pT7Nae.L1926 and pT7Nmo.L2563 in parallel using the s ame p rimers and r un adjacent to the primer extension products as markers. RESULTS Large ORF-containing group IC1 introns in the LSU rDNA from two Naegleria species Screening analyses of LSU rDNA from a number of Naegleria species and lineages revealed large group I introns at two distinct l ocations of the Naegleria sp. isolate NG874 and N. morganensis isolate NG236 [19,22,23]. The 867-bp NG874 intron (named Nae.L1926) is inserted at position 1926 in the LSU rDNA (according to the Escherichia coli LSU rDNA sequence numbering), at the same site as reported in the distantly related protists Rotaliella and Skeletonema [24,25] and only one nucleotide downstream of the well studied nuclear group I introns in Physarum and Tetrahymena [20,26]. Four out of nine analyzed strains of this particular Naegleria species contain almost identical versions of Nae.L1926 [23]. The 940-bp group I intron in N. morganensis (named Nmo.L2563) has the same location in LSU rDNA (position 2563) as introns found in the f ungi Beauveria and Gaeumannomyces [see19]. Nae.L1926, Nmo.L2563 and the Physarum intron Ppo.L1925 (PpLSU3) are the only known nuclear LSU r DNA group I introns harboring ORFs ( Table 1). Secondary structure models of the Naegleria LSU rDNA introns are presented in Fig. 1,A,B, and are based upon known t wo- and three- dimensional features of g roup I intron structures [ 27–31]. These introns are typical group IC1 introns with a structural organization resembling the introns in Physarum and Tetrahymena (Fig. 1C,D). Despite being inserted at differ- ent positions in LSU rDNA, Nae.L1926 and Nmo.L2563 are close relatives sharing about 95% sequence identity in the catalytic core of the group I ribozymes. Nae.L1926 and Nmo.L2563 harbor ORFs as exten sion sequences in the P1 loop segment. ORF-proteins from Nae.L1926 and Nmo.L2563 are members of the His-Cys homing endonuclease family The Nae.L1926 and Nmo.L2563 encoded proteins appear to be 148 and 175 amino acids in size, respectively (Fig. 2A). Both proteins harbor the c onserved His-Cys box motif (Fig. 2 B) present in all nuclear intron homing endonucleases [3,4,6,7,19,32,33], and have been named I-NaeIandI-NmoI. Detailed structural a nd functional analyses o f the related I-PpoI homing endonuclease, encoded by the Ppo.L1925 Ó FEBS 2002 Complex group I introns in Naegleria LSU rDNA (Eur. J. Biochem. 269) 1643 intron, support the hypo thesis that the His-Cys motif is directly involved in the two zinc ion coordination sites [5]. Whereas I-NaeI contains a His-Cys box typical of the two zinc binding motifs, the I-Nm oIseemstolackthemost C-terminal motif. DNA binding and target recognition of I-PpoI have been characterized by biochemical and structural approaches [5,34,35], and revealed an unu sual DNA binding motif consisting of three antiparallel b sheets (b-3, b-4 and b-5). This motif has so far only been recognized in I-PpoIandin the Tn916 integrase [32,36]. Nae.L1926 and Ppo.L1925 introns are located at an almost identical site in the LSU rDNA, suggesting that I-NaeI r ecognizes a nd binds to the same DNA target sequence a s I-PpoI. Interestingly, I-NaeI was found to contain a sequence motif, l ocated approxi- mately 15–40 residues N -terminal of the His-Cys box, with Fig. 1. Secondary structure models of LSU rDNA group IC1 introns.(A,B)Naegleria;(C)Physarum;and(D)Tetrahymena. The paired segments P1–P10 are indicated according to Cech et al . [28]. ORFs are located as P1 extension sequences. I ntron positions are num bered starting with th e first nucleotide of the intron as number 1. Upper case letters represent intron sequences and lowercase letters represent exon sequences. Arrows indicate internal pr ocessing sites (IPS) derived f rom prim er e xtension analysis (PE) or circle ju nction determination (C). Ppo.L 1925 and Tth.L1925 are synonyms for PpLSU3 and TtLSU1, respectively (see Materials and methods). 1644 P. Haugen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 several similarities to the I-Ppo I DNA binding domain (Fig. 2A,C). Critical residues such as Q63 and K65 in b-4 of I-PpoI, known to directly contact the target sequence, are conserved in I-NaeI. Furthermore, structural predictions using the PREDICTPROTEIN and PSIPRED servers (see Mate- rials and methods) support with h igh probability b sheet configurations w ithin t he motif (Fig. 2C). Thes e findings resemble that of the LAGLI-DADG family of homing endonucleases, where residues constituting the DNA bind- ing domain show low sequence conservation among enzymes that recognize the same DNA sequence [37]. The Nae.L1926 and Nmo.L2563 introns self-splice in vitro from their precursor RNAs Group I intron self-splicing proceeds by two sequential trans-esterification reactions resulting in exon ligation and intron excision, and has been well studied in the Tetrahym- ena intron Tth.L1925 (reviewed in [38]) and its cognate Physarum intron Ppo.L1925 [11,20,39,40]. The structural features of Nae.L1926 and Nmo.L2563 (Fig. 1A,B) have significant similarities to Ppo.L1925 and Tth.L1925 (Fig. 1C,D), and we predicted that both the Naegleria LSU rDNA introns self-splice in vitro as naked RNA. To test for self-splicing activity, the corresponding linearized plasmids (see Materials and methods) containing the introns and some flanking exon sequences were transcribed using T7 RNA polymerase, and the corresponding RNAs were subjected to splicing conditions. Representative time course experiments from gel analyses are shown in Fig. 3A. Here, the precursor RNA (RNA 2) and the two products from the self-splicing reaction, e xcised intron ( RNA 3) and ligated exon (RNA 6), can be identified by size. Several additional RNA species appeared on the gels, corresponding to nonligated 5¢ and 3¢ exons (RNAs 8 and 7), circular intron sequences (RNA 1), ORF-containing RNA (RNA 4), a nd free ribozyme (RNA 5). Ligated exons (RNA 6) from both splicing reactions were eluded and purified from the polyacrylamide gels, amplified by RT-PCR and then cloned into plasmid vectors. DNA sequencing of four independent clones from each of the introns confirmed that both Nae.L1926 and Nmo.L2563 excise from their corresponding precursor RNAs and correctly ligate the exons (Fig. 3B). Formation of full-length intron circles is a general feature in the RNA processing of complex group I introns Gel analysis of the Nae.L1926 and Nmo.L2563 splicing reactions (Fig. 3A) indicates that the slow-migrating RNA species (RNA 1) represent i ntron c ircles. T o a nalyze the intron circle junctions, RNA 1a from Nae.L1926 and RNAs 1a and 1b from Nmo.L2563 were eluted from the polyacryl- amide gels, purified and amplified by RT-PCR, and finally cloned into plasmid vectors. DNA sequencing of 10 independent clones showed that RNA 1a from the Nae.L1926 intron corresponds to two equally represented circular species (Fig. 4A); a full-length intron circle (five of 10 clones) and an intron circle lacking the first 15 nucleotides (five of 10 clones). Interestingly, the sequence flanking the )15 circularization site (UGUCUAflAAGAA) is almost identical t o that of the intron 5¢-splice site region (UCU CUUflAAGAA), suggesting that a P1-like structure may be formed prior to the )15 circle formation. The results from the Nmo.L2563 RNA1 are presented in Fig. 4B. Here, the RNA 1a represents full-length intron circles (three of t hree clones). The RNA 1b species migrates slightly slower than the 1.2-kb precursor RNA (Fig. 3A), but consists only of the 389-nucleotide IC1 ribozyme lacking the fi rst 551 nucleotides of the intron (four of four clones; Fig. 4B). The RNA 1b circle resembles the well characterized Tetrahymena intron Tth.L1925 circles lacking 15 or 19 nucleotides (including the exogenous guanosine) at the 5¢ end of the intron [17]. The Ppo.L1925 self-splicing products have previously been reported [20,39,40], but circular intron RNAs were not characterized. I n order to test for Ppo.L1925 circ le forma- tion during self-splicing, linearized p T7Ppo.L1925 plasmid containing the i ntron a nd som e flanking exon sequences were transcribed using T7 RNA polymerase, and t he corresponding RNA was subjected to s plicing conditions for 90 min. The results from g el analysis of the splicing reactions corroborates the findings repo rted previously [20,39], including a slow-migrating RNA species presumed to be a circular RNA (data not shown). By the same experimental approach as described above based on puri- fication, RT-PCR and DNA sequencing, we conclude that Ppo.L1925 generates full-length intron RNA c ircles during incubation in vitro (four of four clones, Fig. 4C). Although full-length intron RNA circles have been rarely reported among the majority of nuclear group I introns studied, all Fig. 2. Sequence features of endonuclease-like O RF-proteins from the Naegleria introns. (A) Primary sequences of I-NaeIandI-NmoI. Putative DNA binding domain and zinc binding motifs (Zn-I and Zn-II) are underlined. (B) Sequence comparison of I-NaeI, I-NmoI, I-PpoI [5] and I-NjaI [33] His-Cys boxes. Conserved zinc coordination residues are enlarge d an d bold. T he asterisk indicates a discont inuity in the seque nce. (C) Structural prediction of a DNA binding motif i n I-NaeI, based on a comparison to crystal stru cture features of I- PpoI [5]. Ide ntical positions are indicated by dots and deletions b y dashes. The DNA binding motif was predicted using the two structural pre- diction s ervers PSIPRED and PREDICTPROTEIN (PHDsec). Secondary structural elements shown are isolated b bridge (B), extended strand (participates in b lad der ; E), 3 10 helix (G), hydrogen bonded turn (T) and bend (S). Ó FEBS 2002 Complex group I introns in Naegleria LSU rDNA (Eur. J. Biochem. 269) 1645 ORF-containing introns analyzed to date generate these circles both in vitro and in vivo (Table 2). Both Naegleria LSU rDNA introns have internal processing sites separating ORF RNAs from the group I ribozymes The splicing reaction of Nae.L1926 and Nmo.L2563, presented in Fig. 3A, generates two major RNA species (RNAs 4 and 5) that could not be explained by the regular splicing pathway. We predicted t hese RNA species to represent the 5¢ half and the 3¢ half of the excised intron, an assumption based on the estimated s ize and on the fact that the similarly organized Ppo.L1925 intron harbours strong internal processing sites separating the ORF RNA from the ribozyme [11,20]. To precisely define the internal processing sites, the Naegleria intron RNAs were incubated under self- splicing conditions for 60 min and subjected to primer extension analysis. The results from the reactions are presented in Fig. 5 and indicate one major processing site located 3¢ of position U469 of Nae.L1926 (Fig. 5A) and two sites 3¢ of positions C549 and C550 of Nmo.L2563 (Fig. 5B). These sites correspond very well to the reported internal processing sites of the Ppo.L1925 intron [11,20] and circularization sites of Tth.L1925 [17], all located close to, or within, the internal guide sequences (see Fig. 1). DISCUSSION We have characterized in vitro RNA processing of two Naegleria group I introns, Nae.L1926 and Nae.L2563, both harbouring ORFs within the L1-loop of group IC1 ribo- zymes. The intron ORFs correspond to His- Cys h o ming endonucleases and are named I-NaeIandI-NmoI, respect- ively. I-Na eI has a motif similar to the antiparallel b sheet DNA binding domain found in I-PpoI. Whereas almost a ll His-Cys homing endonucleases have two zinc coordination domains, the C-terminal domain appears to be missing in I-NmoI. In vitro analyses show that both introns self-splice, generate full-length RNA circles, and harbour internal Fig. 3. Gel analysis of the in vitro self-spli- cing products of Nae.L1926 and Nmo.L2563. (A) RNA was incubated at self-splicing conditions for 0–30 min and analysed on an 8 M urea/5% polyacrylamide gel. The observed RNAs after 30 min incubation are full-length intron circles (RNA 1a), circles containing only the group IC1 ribozyme (RNA 1b), precursor (RNA 2), excised intron (RNA 3), intron ORF (RNA 4), intron ribozyme (RNA 5), ligated exon (RNA 6), free 3¢ exon (RNA 7), and fre e 5¢ exon (RNA 8). M, RNA size marker. The 3¢ exon RNA of Nmo.L2563 was run off the gel. (B) Sequencing ladder of amplified ligated exon generated from Nae.L1926 and Nae.L2563 intron splicing. The RNA was purified from a gel, subjected to R T-PCR amplification, plasmid cloned and sequenced. The corresponding ligated exon RNA sequences are p resented below. Arrows indicate exon junctions. 1646 P. Haugen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 processing sites close to, or at, their internal guide sequences. Full-length intron circles In organisms like Naegleria, group I intron splicing is an essential reaction to the host in order to generate functional rRNAs. However, intron processing reactions like internal cleaving, 3¢ SS hydrolysis and intron circularization are more likely to be selfish features of the nucleolar group I introns. A ll ORF-containing or complex n uclear group I introns tested so far generate full-length intron RNA circles in viv o or when incubated at self-splicing conditions in v itro (Table 2). The biological role of full-length circularization of intron RNA is not clear, but one possibility is a s a n intermediate in endonuclease expression. I-PpoI is reported to be expressed from the full-length intron and not from the internally process ed RNA [15], an observation consistent with the idea that full-length intron circles might be involved. Because I-PpoI mRNA is not polyadenylated in vivo [14] a circular RNA could increase the stability, or translocation from the nucleus to the cytoplasm, prior to translation. Alternatively, full-length intron circles may be involved in intron horizontal transfer at the RNA level [21,41,42]. Internal processing of intron RNA There are strong links b etween internal intron RNA processing and the expression of nuclear group I intron homing endonucleases. Functional studies both in vitro and in vivo of twin-ribozyme group I introns in Didymium and Naegleria im plies that i nternal p rocessing, catalysed by a second internal group I-like ribozyme, is an essential step in the expression of the corresponding homing endonuclease genes [12,13,43–45]. In vitro studies of the Ppo.L1925 intron mapped a n internal processing site 53 nucl eotides down- stream of the I- PpoI ORF stop codon, proximal to the internal guide sequence o f the splicing ribozyme [20]. Analyses in yeast s how that I- PpoI i s expressed f rom an RNA polymerase I transcribed full-length intron RNA, but not from the internal processed RNA [11,15]. Thus, in contrast to the twin-ribozyme intron the internal processing of Ppo.L1925 intron RNA appears to down-regulate endonuclease expression. The Naegleria LSU rDNA introns have several features in common to Ppo.L1925. They have all large insertions at the s ame l ocation in P 1 (L1-loop) within their g roup IC1 ribozyme s tructures. The L 1-loop Fig. 5. Mapping of the internal processing sites. (A) Nae.L1926 and (B) Nmo.L2563. Primer e xtension p roducts (PE) generated f rom s elf- spliced Nae.L1926 and Nmo.L2563 intron RNAs were analysed together with the corresponding DNA s equence marker. The DNA sequence is complementary to the RNA sequence shown in the lower panels. Processing sites are indicated by arrows. The internal guide sequences (IGS) are underlined. Fig. 4. Analysis of intron RNA circle junctions. from (A) Nae.L1926 (B) Nmo.L2563 and (C) Ppo.L1925. Regions correspon ding to circle junctions of isolated intron RNAs were amplified by RT-PCR and sequenced. Circle junctions are indicated 3¢ to the last residue of the intron (xG). RNA sequences of junctions corresponding to full-length intron circles (FL), )15 nucleotide circles ()15), and )551 nucleotide circles ()551) are presented in the lower panels. Ó FEBS 2002 Complex group I introns in Naegleria LSU rDNA (Eur. J. Biochem. 269) 1647 extension sequences contain ORFs with the characteristic histidine a nd cysteine motifs common among al l nuclear homing endonucleases [3]. Finally, Ppo.L1925, Nae.L1926 and Nmo.L2563 generate full-length intron RNA circles as well as internal processing sites at ap proximately the same positions within the ribozyme structure. These similarities in sequence, organization and in vitro processing might indicate a similar biological role in down-regulation of endonuclease expression of the internal processing sites. However, Nae.L1926 and Nmo.L2563 differ significantly from Ppo.L1925 in several important aspects. Although the I-NaeIandI-NmoI ORFs appear unrelated in primary sequence, sequence similarities are present (Fig. 6) at the 5¢ and 3¢ untranslated regions. Here, the Naegleria 3¢ untrans- lated regions are only 13 nucleotides compared to the corresponding 53-nucleotide structured region in Ppo.L1925 [14]. Whereas the I-PpoI RNA harbours no polyadenylation signal and seems not to be polyadenylated in vivo [14], both Naegleria introns contain the AAUAAA consensus poly- adenylation signal located exactly 12 and 28 nucleotides upstream of the stop codons (UAG/UAA) and internal processing sites, respectively (Fig. 6). 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(2000) Molecular phylogeny of the family Ankist rode sma ceae (Chlorophy ta, Chlorophycea e) and ana lyse s of their group I intervening sequences, PhD Thesis, University of Erlangen, Germany. 48. Decatur, W.A., Einvik, C., Johansen, S. & Vogt, V.M. (1995) Two group I ribozymes with different functions in a nuclear rDNA intron. EMBO J. 14, 4558–4568. Ó FEBS 2002 Complex group I introns in Naegleria LSU rDNA (Eur. J. Biochem. 269) 1649 . Characterization of the self-splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes Peik Haugen 1 ,. 2002 some intron systems that include the ORF -containing group I introns (Table 2). In vivo analyses of I- PpoI, I- DirI and I- NgrI expression in their original