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

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

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.

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.

(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

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

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-

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

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)

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

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.

internal processing stimulates endonu clease expression.
ACKNOWLEDGEMENT
This work was supported by grants to S. J. from T he Norwegian
Research Council, The Norwegian Cancer Society, and The Aakre
Foundation for Cancer Research.
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