De Jonckheere2and Steinar Johansen1 1 RNA Research group, Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway;2Protozoology Laborato
Trang 1Characterization of the self-splicing products of two complex
genes
Peik Haugen1, Johan F De Jonckheere2and Steinar Johansen1
1
RNA Research group, Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway;2Protozoology 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
self-splice in vitro, generating ligated exons and full-length intron
circles as well as internal processed excised intron RNAs
Formation of full-length intron circles 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-NaeI and I-NmoI encoded by Nae.L1926 and Nmo.L2563, respectively, correspond to His-Cys homing endonucleases of 148 and 175 amino acids I-NaeI contains
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 the Ppo.L1925 intron ORF
Keywords: group I ribozyme; mobile intron; ribosomal DNA; RNA processing
About 3% of the 850 nuclear group I introns 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 role 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 role of group I intron endonucleases
appears to be in intron homing at the DNA level [7]
Homing is initiated by a double-strand break made by the
endonuclease at an intron-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-PpoI and I-DirI from nuclear group I
introns Ppo.L1925 and Dir.S956-1 in the myxomycetes
Physarum polycephalum and Didymium 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 RNA polymerase I transcribed precursor rRNA An intriguing question is thus how intron proteins encoded by nuclear group I introns are expressed from an RNA polymerase I transcript A protein encoding gene in a eukaryotic nucleus is in general transcribed by RNA polymerase II as premRNA Here, pre-mRNA maturation 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 strategies 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], and sequences downstream the I-PpoI ORF RNA, preceding the group I ribozyme, were found to be important in both splicing and 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 associated I-DirI mRNA supports 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, with a subsequent ligation of flanking exon sequences [17] Additional ribozyme-cata-lyzed RNA processing reactions, including intron circular-ization and internal processing, have been 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)
Trang 2some 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-NaeI and I-NmoI, respectively In order to gain insight into cellular maturation and processing patterns of the I-NaeI and I-NmoI mRNAs, we analysed their corresponding intron RNAs for splicing and self-processing in vitro
M A T E R I A L S A N D M E T H O D S
Plasmid construction, DNA sequencing, and computer analyses
Introns are named according to the new proposed nomen-clature of 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 rDNA segments using the primer sets OP460 (5¢-AATTAATACGACTCACTATAGGTCC TGCACACCTTGT-3¢)/OP461 (5¢-CGCCAGACTAGAG TCA-3¢), OP454 (5¢-AATTAATACGACTCACTATAGG CGGATAAGGCCAAT-3)/OP451 (5¢-GCTCACGTTCC CTGT-3¢), and OP452 (5¢-AATTAATACGACTCACTAT 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 pUC18 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.
a
Named according to [18].bPutative 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
exonsa
Full-length circles b
In vitro/
in vivo
Internal processing sites c
in vitro/
in vivo Reference
Dir.S956-1 + +/+ +/+ [12,44,48]
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 anaribozyme-cata-lyzed
(NA).
Trang 3Biotech), yielding pT7Nae.L1926, pT7Nmo.L2563, and
pT7Ppo.L1925, respectively The inserts were sequenced
using the Thermo Sequenase sequencing kit (Amersham
Pharmacia Biotech) and [a-33P]ddNTPs (GATC;
450 lCiÆmL)1) The Nmo.L2563 intron was 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 theGCGsoftware 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 templates pT7Nae.L1926
(BamHI), pT7Nmo.L2563 (HindIII), and pT7Ppo.L1925
(HindIII) The RNA was uniformly labelled using
[a-35S]CTP (10 lCiÆlL)1; Amersham Pharmacia Biotech),
and subjected to self-splicing conditions (40 mM Tris
pH 7.5, 0.2MKCl, 2 mMspermidine, 5 mMdithiothreithol,
10 mMMgCl2and 0.2 mMGTP) at 50°C for 0–30 min, all
essentially as described previously [1] Self-spliced RNA was
subjected to electrophoresis in a 5% polyacrylamide, 8M
urea gel, and visualized by autoradiography
RNA circle and exon junction determination
Ligated exon and circular intron RNAs were isolated from
polyacrylamide gels and incubated in 400 lL elution buffer
(0.3MNH4Ac, 0.1% SDS, 10 mMTris pH 8 and 2.5 mM
EDTA pH 8) on a rotating wheel at 4°C over night The
RNA was purified using a 0.45-lM filter (Millipore), and
ethanol precipitated PAGE-purified RNA was
subse-quently subjected to reverse transcription using the First
Strand cDNA Synthesis kit (Amersham Pharmacia
Biotech) and a downstream primer Products were amplified
by adding an upstream primer, 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 the 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 full-length intron RNA
circle was analysed by using the primer set OP444
(5¢-GGGTG CAGTTCACAGACT-3¢)/OP443 (5¢-ATGG
TACATGGT GCGTTA-3¢)
Mapping of internal processing sites
The 5¢ ends of the internal processing sites were 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 RNA was subse-quently purified in several steps including phenol/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 and OP558 The reverse transcrip-tion reactranscrip-tions were performed using the SuperScript II (Gibco BRL) enzyme with 10 lCi [a-35S]dCTP (Amersham Pharmacia Biotech) as the label DNA sequencing ladders were prepared from pT7Nae.L1926 and pT7Nmo.L2563 in parallel using the same primers and run adjacent to the primer extension products as markers
R E S U L T S
Large ORF-containing group IC1 introns
in the LSU rDNA from twoNaegleria species Screening analyses of LSU rDNA from a number of Naegleriaspecies and lineages revealed large group I introns
at two distinct locations 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 fungi Beauveria and Gaeumannomyces [see19] Nae.L1926, Nmo.L2563 and the Physarum intron Ppo.L1925 (PpLSU3) are the only known nuclear LSU rDNA 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 two- and three-dimensional features of group 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 extension 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 conserved His-Cys box motif (Fig 2B) present in all nuclear intron homing endonucleases [3,4,6,7,19,32,33], and have been named I-NaeI and I-NmoI Detailed structural and functional analyses of the related I-PpoI homing endonuclease, encoded by the Ppo.L1925
Trang 4intron, support the hypothesis 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-NmoI seems to lack the most
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 unusual 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-PpoI and in 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 recognizes and binds to the same DNA target sequence as I-PpoI Interestingly, I-NaeI was found to contain a sequence motif, located 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 Intron positions are numbered starting with the first nucleotide of the intron as number 1 Upper case letters represent intron sequences and lowercase letters represent exon sequences Arrows indicate internal processing sites (IPS) derived from primer extension analysis (PE) or circle junction determination (C) Ppo.L1925 and Tth.L1925 are synonyms for PpLSU3 and TtLSU1, respectively (see Materials and methods).
Trang 5several similarities to the I-PpoI 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 thePREDICTPROTEINand PSIPREDservers (see
Mate-rials and methods) support with high probability b sheet
configurations within the motif (Fig 2C) These 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, excised 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), and 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 intron circles To analyze 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 to 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 three 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 first 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 In order to test for Ppo.L1925 circle forma-tion during self-splicing, linearized pT7Ppo.L1925 plasmid containing the intron and some flanking exon sequences were transcribed using T7 RNA polymerase, and the corresponding RNA was subjected to splicing conditions for 90 min The results from gel analysis of the splicing reactions corroborates the findings reported 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 circles 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 ORF-proteins from the
Naegleria introns (A) Primary sequences of I-NaeI and I-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 enlarged and bold The asterisk indicates a discontinuity in
the sequence (C) Structural prediction of a DNA binding motif in
I-NaeI, based on a comparison to crystal structure features of I-PpoI
[5] Identical positions are indicated by dots and deletions by dashes.
The DNA binding motif was predicted using the two structural
pre-diction servers PSIPRED and PREDICTPROTEIN (PHDsec) Secondary
structural elements shown are isolated b bridge (B), extended strand
(participates in b ladder; E), 3 10 helix (G), hydrogen bonded turn (T)
and bend (S).
Trang 6ORF-containing introns analyzed to date generate these
circles both in vitro and in vivo (Table 2)
BothNaegleria 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 these RNA species to
represent the 5¢ half and the 3¢ half of the excised intron, an
assumption based on the estimated size 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)
D I S C U S S I O N
We have characterized in vitro RNA processing of two Naegleriagroup 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 homing endonucleases and are named I-NaeI and I-NmoI, respect-ively I-NaeI has a motif similar to the antiparallel b sheet DNA binding domain found in I-PpoI Whereas almost all 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 free 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 RT-PCR amplification, plasmid cloned and sequenced The corresponding ligated exon RNA sequences are presented below Arrows indicate exon junctions.
Trang 7processing 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 All ORF-containing or complex nuclear group I
introns tested so far generate full-length intron RNA circles
in vivoor when incubated at self-splicing conditions in vitro (Table 2) The biological role of full-length circularization
of intron RNA is not clear, but one possibility is as an intermediate in endonuclease expression I-PpoI is reported
to be expressed from the full-length intron and not from the internally processed 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 between internal intron RNA processing and the expression of nuclear group I intron homing endonucleases Functional studies both in vitro and
in vivoof twin-ribozyme group I introns in Didymium and Naegleria implies that internal processing, 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 an internal processing site 53 nucleotides down-stream of the I-PpoI ORF stop codon, proximal to the internal guide sequence of the splicing ribozyme [20] Analyses in yeast show that I-PpoI is expressed from 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 same location in P1 (L1-loop) within their group IC1 ribozyme structures The L1-loop
Fig 5 Mapping of the internal processing sites (A) Nae.L1926 and (B)
Nmo.L2563 Primer extension products (PE) generated from
self-spliced Nae.L1926 and Nmo.L2563 intron RNAs were analysed
together with the corresponding DNA sequence 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 corresponding 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.
Trang 8extension sequences contain ORFs with the characteristic
histidine and cysteine motifs common among all 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 approximately 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-NaeI and I-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
Naegleriaintrons 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) Polyadenylation of
homing endonuclease mRNAs has been reported in two
different nuclear group I introns in Didymium [12,46] Both
I-DirI and I-DirII mRNAs contain AAUAAA
polyadeny-lation signals 15 nucleotides upstream of the
polyadeny-lation tails These observations suggest that both the I-NaeI
and I-NmoI mRNAs appear polyadenylated in vivo,
prob-ably at their internal processing sites, and implies that
internal processing stimulates endonuclease expression
A C K N O W L E D G E M E N T
This work was supported by grants to S J from The Norwegian
Research Council, The Norwegian Cancer Society, and The Aakre
Foundation for Cancer Research.
R E F E R E N C E S
1 Haugen, P., Huss, V.A., Nielsen, H & Johansen, S (1999)
Complex group-I introns in nuclear SSU rDNA of red and green
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