The group I-like ribozyme DiGIR1 mediates alternative processing
of pre-rRNA transcripts in
Didymium iridis
Anna Vader
1,2
, Steinar Johansen
2
and Henrik Nielsen
1
1
Department of Medical Biochemistry and Genetics, The Panum Institute, Copenhagen, Denmark;
2
Department of Molecular
Biotechnology, Institute of Medical Biology, University of Tromsø, Norway
During starvation induced encystment, cells of the myxo-
mycete Didymium iridis accumulate a 7.5-kb RNA that is the
result of alternative processing of pre-rRNA. The 5¢ end
corresponds to an internal processing site cleaved by the
group I-like ribozyme DiGIR1, located within the twin-
ribozyme intron Dir.S956-1. The RNA retains the majority
of Dir.S956-1 including the homing endonuclease gene and a
small spliceosomal intron, the internal transcribed spacers
ITS1 and ITS2, and the large subunit rRNA lacking its two
group I introns. The formation of this RNA implies clea-
vage by DiGIR1 in a new RNA context, and presents a new
example of the cost to the host of intron load. This is because
the formation of the 7.5-kb RNA is incompatible with the
formation of functional ribosomal RNA from the same
transcript. In the formation of the 7.5-kb RNA, DiGIR1
catalysed cleavage takes place without prior splicing per-
formed by DiGIR2. This contrasts with the processing order

splice site hydrolysis activity, which induces the formation
of full-length intron RNA circles using a processing
pathway that is distinctly different from splicing ([5];
unpublished data]. The other ribozyme (DiGIR1), which
along with the I-DirI HEG is inserted in DiGIR2, carries
out hydrolysis at two internal processing sites (IPS1 and
IPS2) located at its 3¢ end [5,6]. In vivo, this cleavage results
in the formation of the 5¢ end of the I-DirImRNAandis
followed by cleavage at an in vivo specific internal processing
site (IPS3) downstream of the HEG and by polyadenylation
(summarized in Fig. 1, left panel). Finally, a 51-nucleotide
spliceosomal intron (I51) within the HEG RNA is removed
before the resulting I-DirI mRNA is transported to the
cytoplasm where it associates with the polysomes [7].
Homing activity of the I-DirI protein has been demonstra-
ted by Dir.S956-1 intron mobility studies involving genetic
crosses between intron-containing and intron-lacking
Didymium isolates [8].
During our work on the in vivo expression of Dir.S956-1,
we noted the presence of an I-DirI HEG-containing RNA
species that migrated similarly to the 7.46-kb ladder band on
a denaturing agarose gel, but did not hybridize to an SSU
probe. This observation, as well as reverse transcription/
PCR analyses which showed that Dir.S956-1 produces full-
length intron circles in vitro [9] and in vivo [10], led us to
believe that this unknown RNA represented a circular
species that was retarded in the gel during electrophoresis.
We have subsequently observed that the signal intensity of
the 7.5-kb band varies greatly according to the state of the
D. iridis culture when the RNA was isolated. To address the

)1
MgSO
4
,1mgÆmL
)1
KH
2
PO
4
,1.5mgÆmL
)1
K
2
HPO
4
) containing Escherichia coli cells. Cells and cysts
were counted in a Tu
¨
rk chamber or electronically in a
Coulter Multisizer (Coulter Electronics Ltd). Cysts were
scored by their ability to resist lysis in 0.5% Nonidet P-40
[11]. Cysts were stained by the addition of 1 vol. 0.25%
Trypan Blue in standard NaCl/P
i
.
For RNA extraction, a total of 10
7
Didymium cells
were harvested by centrifugation at 400 g for 5 min The
pellet was dissolved in 1 mL Trizol Reagent (Gibco-BRL)

Fig. 1. Homing endonuclease gene expression and life cycle of D. iridis. Proposed processing pathways in the formation of RNA species encoded by
the Dir.S956-1 homing endonuclease gene (HEG). In vegetatively growing D. iridis, the Dir.S956-1 intron is spliced out from pre-rRNA and further
processed into an I-DirI endonuclease mRNA (left panel; see [7] for details). Starvation/encystment results in an alternative processing pathway of
the intron (right panel), induced by DiGIR1 ribozyme cleavage at an internal intron processing site. Subsequently, a 7.5-kb linear RNA is formed
after the excision of the LSU rRNA introns Dir.L1949 and Dir.L2449. The accumulated 7.5-kb RNA contains all of the Dir.S956-1 sequences
except those encoding the cleavage ribozyme DiGIR1. A possible functional role of the 7.5-kb RNA is as an alternative precursor for the
endonuclease mRNA during excystment. Here, the HEG RNA might be separated from the remaining 7.5-kb RNA sequences by cleavage at the
host induced IPS3 or the ribozyme induced 3¢ splice site. (A) 1.46 kb RNA (the full length intron after splicing). (B) 1.23 kb RNA (resulting from
GIR1 cleavage). (C) 0.90 kb RNA (resulting from cleavage at IPS3). (D, E) Nuclear and cytoplasmic form of the 0.85 kb RNA also referred to as
the I-DirI mRNA. (Inset) Life cycle of the myxomycete D. iridis. Haploid amoebae or swarm cells can transform into dormant cysts under
unfavourable environmental conditions. This process is reversible. Alternatively, two compatible amoebae or swarm cells can act as gametes and
fuse to produce a diploid zygote. Growth of the zygote is accompanied by a series of nuclear divisions, leading to the formation of a multinucleated
plasmodium. Eventually, the plasmodium transforms into fruiting bodies, which release haploid spores. Germination of the spores completes the
life cycle, in that vegetative amoebae or swarm cells are formed again.
Ó FEBS 2002 Ribozyme mediated processing of pre-rRNA (Eur. J. Biochem. 269) 5805
GTTCAGAGACTATA-3¢;OP169,5¢-ACCTAAGGC
GGACGTTACTG-3¢;OP180,5¢-GCCTCCCTTGGGA
TAT-3¢; OP448, 5¢-AACCGAACAATGAGACTGAA-3¢;
OP449, 5¢-CTCGTATTCGAAGGCATGCA-3¢;C78,
5¢-TGCTTCCTTTCGGAACGA-3¢; C231, 5¢-ATTCCGA
TATCGTGCTCTA-3¢; C232, 5¢-AAGAGGTTGGCCAA
GGAA-3¢; SSU6, 5¢-CGAATTCAGGGGCAACATCGG
TTC-3¢;SSU7,5¢-CGAATTCACCGAGGTTACAAG
GCA. The ETS, GIR1, HEG, GIR2 and LSU1 PCR
products were purified on S-300 spin-columns (Pharmacia)
prior to labelling by random priming using the Mega Prime
kit (Amersham) and [a-
32
P]dCTP (3000 CiÆmmol
)1

heated in 1 · RNaseH buffer (GibcoBRL) at 80 °Cfor
1min.At45°C, 20 U RNasin (Pharmacia) was added, and
the sample incubated for 10 min. After transfer to ice, 0.5 U
RNaseH (GibcoBRL) was added to produce a total volume
of 10 lL. The sample was then incubated at 30 °Cfor
5 min, prior to analysis by Northern blotting (see above).
Primer extension
For primer extension, gel-purified OP4 was labelled with
[a-
32
P]ATP (3000 CiÆmmol
)1
, Amersham) using T4 poly-
nucleotide kinase (Gibco-BRL). RNA was added to 2 pmol
labelled oligo in 1 · RT buffer (50 m
M
Tris/HCl at pH 8,
60 m
M
KCl, 10 m
M
MgCl
2
,1m
M
dithiothreitol) in a total
volume of 5 lL, denatured at 80 °Cfor2minand
incubated at 45 °C for 10 min. Subsequently 4 lLRNA/
oligo mixture was added to a tube containing 1 U AMV
reverse transcriptase (RT; Pharmacia), 1 U RNasin

M
CaCl
2
,0.1m
M
EDTA,
0.16 m
M
cycloheximide, 0.5% Nonidet P-40, 500 UÆmL
)1
RNasin), incubated for 5 min in ice/water to allow lysis of
the cells and centrifuged at 10 000 g,4°C for 10 min. The
pelleted nuclei were dissolved in Trizol (nuclear RNA), and
the supernatant was extracted with phenol/chloroform and
precipitated by EtOH (cytosolic RNA).
For sucrose gradients, 250 lg whole cell RNA was
heated to 70 °C for 5 min, cooled on ice and centrifuged at
13 000 g,4°C for 5 min The supernatant was loaded onto
a linear 15–40% sucrose gradient in 10 m
M
Tris/HCl at
pH 7.5, 100 m
M
LiCl, 10 m
M
EDTA and 0.2% SDS and
centrifuged for 20 h at 4 °C and 25 000 r.p.m. in a Beckman
SW27.1 rotor. Fractions of approximately 1 mL were
collected and RNA was isolated by phenol/chloroform
extraction.

mind that cyst formation is most likely committed
biochemically long before this time. Examination of whole
cell RNA from a time course of a Didymium culture shows
that the 7.5-kb RNA is hardly detectable at the first time
points when food is plentiful, but becomes abundant when
the cells are starved and the culture reaches the stationary
phase (Fig. 2B). At the last time points the 7.5-kb RNA is
the predominant HEG RNA in the cells. While the
amounts of some of the other HEG RNA species also
vary, none exhibits the same pattern. It is interesting to
note that another prominent signal corresponding to a 3.9-
kb RNA, which comigrates with the LSU rRNA, decrea-
ses as the 7.5 kb signal increases. The 3.9 kb RNA appears
5806 A. Vader et al. (Eur. J. Biochem. 269) Ó FEBS 2002
to be a nuclear species [7], and a similar RNA has been
observed when whole cell RNA from the Didymium CR8
isolate was probed with the Dir.S956-2 group I intron [10].
The fact that the Dir.S956-1 and Dir.S956-2 group I
introns are unrelated [10], suggests that the formation of
the 3.9 kb RNA is independent of the intron, and results
from a more general alternative pathway of Didymium pre-
rRNA processing.
The 7.5-kb signal is a linear RNA made by alternative
processing of the pre-rRNA
To confirm that the 7.5-kb signal indeed represented a
circular form of the Dir.S956-1 intron RNA, the following
experiments were carried out. First, RNA from the time
course experiment shown in Fig. 2, was analysed on a
denaturing 4% polyacrylamide gel in diluted electro-
phoresis buffer (0.4 · TBE). Under these conditions we

an alternative processing of the pre-rRNA in which the SSU
rRNA sequence upstream of Dir.S956-1 is removed. This
hypothesis would similarly be consistent with the previously
published observation of lack of hybridization of an
upstream SSU probe to the 7.5-kb RNA [7]. Considering
the low abundance of this RNA compared with ribosomal
RNAspecies,andthefactthatitcoexistswithRNAs
containing the same structural elements, we decided to
deduce its structure by analysis of preparations of whole cell
RNA rather than to isolate it. Whole cell RNA was isolated
from Didymium cellsharvestedearlyandlateinatime
course (corresponding to time points 1 and 6 in Fig. 2).
These RNAs were analysed by Northern blotting and
RNaseH cleavage. In the Northern blotting analysis,
parallel filters were hybridized with a panel of probes
complementary to different parts of the Didymium pre-
rRNA including ETS, HEG, ITS1 and ITS2 (Fig. 3A). A
signal of 9.5 kb was detected only by the non-Dir.S956-1
probes (i.e. ETS, ITS1 and ITS2; Fig. 3B), suggesting that it
represents the pre-rRNA subsequent to Dir.S956-1 excision.
The observation of the 9.5 kb RNA is in agreement with
splicing being one of the earliest events in pre-rRNA
processing, as previously shown for the Tth.L1925 intron in
Tetrahymena thermophila [12]. Although information on the
precise location of the 5¢ and 3¢ ends of Didymium pre-
rRNA is not available, the size of 9.5 kb for this RNA is
in reasonable agreement with the expected size based
on reported ribosomal DNA sequences from different
Didymium isolates.
Fig. 3. Characterization of the 7.5-kb RNA signal. (A) Schematic presentation of the D. iridis (Lat3-5 strain) rDNA. The upper panel shows the

In the RNaseH analysis, the parallel RNA samples
were hybridized to oligonucleotides complementary to
different parts of the Didymium pre-rRNA (see Fig. 3A).
RNaseH, which will degrade the RNA strand of the
resulting RNA : DNA heteroduplexes, was then added.
The resulting RNA fragments were visualized by
Northern blotting analysis using a HEG probe. The
experiment showed that oligonucleotides complementary
to ITS-1, ITS-2, LSU as well as the SSU-sequence
downstream of Dir.S956-1 will induce RNaseH-catalysed
cleavage of the 7.5-kb RNA and result in cleavage
products of the expected size (Fig. 3C). Oligonucleotides
complementary to SSU sequences upstream of Dir.S956-1,
on the other hand, had no effect (data not shown),
showing that these sequences are not part of the 7.5-kb
RNA. Taken together, the data from Northern blotting
and RNaseH analyses are consistent with the hypothesis
that the 7.5-kb RNA signal is a linear species produced by
alternative processing of the pre-rRNA.
Only parts of the Dir.S956-1 intron are included in the
7.5-kb RNA
To determine if all of the Dir.S956-1 intron is included in the
7.5-kb RNA, filters containing whole cell RNA from early
and late points in a time course (see above) were hybridized
with probes complementary to GIR1, HEG and GIR2
(Fig. 4A). Surprisingly, the Northern blotting results dem-
onstrated that GIR1 is absent from the 7.5-kb RNA
(Fig. 4B). RNaseH analyses substantiated this conclusion in
that oligonucleotides complementary to GIR1 did not
cleave the 7.5-kb RNA, whereas oligonucleotides that

position of the 5¢ and 3¢ splice sites (SS) as well as the internal pro-
cessing sites (IPS). The exon, open reading frame and intron sequences
are shown in black, grey and white, respectively. The localization of the
probes used for Northern blotting analysis are indicated with thick
black lines. (B) Northern blotting analysis of whole cell RNA. Parallel
filters containing RNA from an early (E) and a late (L) time point
(corresponding to positions 1 and 6 in Fig. 2A) were hybridized with
the indicated GIR1, HEG and GIR2 probes. The G319 RNA marker
(Promega) was used as a size marker.
Ó FEBS 2002 Ribozyme mediated processing of pre-rRNA (Eur. J. Biochem. 269) 5809
cells. As expected, IPS2-terminated RNAs were found in
the low molecular mass fractions where the processed
forms of the excised Dir.S956-1 (1.23-kb RNA and 0.85-
kb RNA; see Fig. 2) are located. Another IPS2-termin-
ated HEG RNA exists in the high molecular fractions
where the 7.5-kb RNA is the predominant HEG RNA.
This implies that the 5¢ end of the 7.5-kb RNA
corresponds to IPS2. While DiGIR1 cleaves at two
processing sites (IPS1 and IPS2) in an obligate sequential
order in vitro [6], only IPS2-cleaved RNA has been
detected in vivo [7]. We infer that 5¢ end formation of the
7.5-kb RNA is a result of DiGIR1 catalysis. The critical
involvement of DiGIR1 in the formation of the 5¢ end of
the RNA could be tested by the introduction of mutations
in the catalytic site of the ribozyme [3]. Unfortunately, a
transformation protocol for Didymium is currently not
available.
The 7.5-kb RNA is located in the nucleus
Previous studies have shown that the different I-DirIHEG
RNAs differ in their intracellular distribution. While the

5
Lat3-5 cells was run on a 1%
denaturing agarose gel and analysed by hybridization using the HEG
probe described in Fig. 3A. The identity of the observed signals is
indicated on the right. The size indications are derived from the High
Range RNA ladder (Fermentas).
Fig. 5. Mapping of the 5¢ end of the linear 7.5-kb RNA. Whole cell
RNA (250 lg) from a late time point (corresponding to position 6 in
Fig. 2A) was fractionated on a 15–40% sucrose gradient. In addition
to the pelleted material that had run through the gradient (P), 22
fractions were collected (1–22). (A) Denaturing agarose gel of fract-
ionated RNA. RNA was recovered from the collected fractions and
analysed on a 1% agarose gel stained with ethidium bromide. The
positions of the SSU and LSU rRNAs are indicated. The 0.24- to 9.5-
kb ladder (GibcoBRL) was used as a size marker (L). (B) Northern
blotting analysis of fractionated RNA. The gel shown in (A) was
analysed by hybridization using the HEG probe indicated in Fig. 4A.
The positions of the 7.5-kb RNA and the processed forms of the intron
(1.46-kb, 1.23-kb, and 0.85-kb RNA, respectively) are shown. The size
indications are derived from the ladder shown in (A). (C) Primer
extension analysis of fractionated RNA. RNA recovered from the
collected fractions was analysed using OP4 (an 18-mer complementary
to a sequence 35–52 nucleotides downstream of IPS2) as a primer. OP4
wasalsousedtomakeaDNAsequencingladderwhichwasusedto
determine the exact position of the primer extension stop at IPS2 as
indicated.
5810 A. Vader et al. (Eur. J. Biochem. 269) Ó FEBS 2002
contains all sequence elements downstream of IPS2 (see
Fig. 1, right panel). These include the LSU rRNA exons,
the internal transcribed spacers (ITS1 and ITS2) and the

The composition of the 7.5-kb RNA suggests how it is
formed and no new activities need to be postulated to
account for its structure. Instead, its accumulation can be
explained by an alteration of the relative rates of known
processing activities. All of the experiments carried out in
the present study aim at analysing the steady-state level of
the RNAs involved. It is frequently observed that the
processing of pre-rRNA and pre-mRNA slows down
when cells are starved. In the ciliate Tetrahymena pyrifor-
mis, the pre-rRNA processing rate has been reported to be
decreased 36-fold during starvation [13] and a similar
12-fold decrease has been observed in T. thermophila [14].
As a result, precursors and processing intermediates
tend to show increased steady-state levels under such
conditions. In the present case, this could explain the
inclusion of ITS1 and ITS2 in the 7.5-kb RNA, as well as
the presence of the spliceosomal intron and the failure to
use the polyadenylation signals. The observed nuclear
localization of the 7.5-kb RNA is expected as the RNA
retains several putative nuclear retention signals, e.g. a
spliceosomal intron.
The accumulation of the 7.5-kb RNA during starva-
tion does not necessarily imply that this RNA is specific
for starved cells. It is possible that the 7.5-kb RNA is a
default intermediate in the formation of the I-DirI
mRNA that is rapidly turned over during normal
exponential growth and thus not detected by the methods
applied in the present study. This would leave the excised
intron as a dead end rather than as a precursor in the
formation of I-DirI mRNA. We are currently unable to

to occur even in the presence of 300 lgÆmL
)1
actinomy-
cin D, suggesting that new RNA synthesis is not required
[16]. Rather, stable messengers are stored by the cysts in
preparation for rapid emergence. Protein synthesis seems
to be required for excystment [11,16], although these
findings have been disputed [17]. Although it is quite
possible that the 7.5-kb RNA is a dead-end product, it
remains a possibility that it functions as a precursor for
the I-DirI mRNA during excystment. Preliminary experi-
ments aimed at following the 7.5-kb RNA through
germination of cysts have failed due to lack of synchrony
in cultures of germinating cysts.
In conclusion, we have described a new RNA species that
accumulates by alternative processing of pre-rRNA during
starvation-induced encystment in Didymium.Themost
interesting aspect of this RNA is perhaps the implications of
its formation. It provides a new example of the cost of
intron load on the host cell because the formation of this
RNA is incompatible with the formation of ribosomal
RNA from the same transcript. It shows DiGIR1 activity in
a different RNA context than previously demonstrated and
shows that the activities of the splicing and cleavage
ribozymes of a twin ribozyme intron can be modulated
with respect to each other resulting in different processing
products.
ACKNOWLEDGEMENTS
We thank F. Frenzel for technical assistance and J. Christiansen for
helpful suggestions to the experiments. This work was supported by

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