Ribozyme DiGIR1: Báo cáo Y học về vai trò trung gian xử lý RNA tiền thân thay thế ở Didymium iridis

The group I-like ribozyme DiGIR1 mediates alternative processing

of pre-rRNA transcripts in

Didymium iridis

Anna Vader

1,2

, Steinar Johansen

and Henrik Nielsen

Department of Medical Biochemistry and Genetics, The Panum Institute, Copenhagen, Denmark;

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

leading to mature rRNA and I-DirI mRNA in growing cells,

suggesting an interplay between the two ribozymes of a twin-

ribozyme intron.

Keywords:Didymium iridis; group I intron; ribozyme; pre-

rRNA processing.

Group I introns contain a conserved set of sequences and

structural elements that are involved in the removal of the

intron by splicing. They constitute one class out of fewer

than 10 classes of naturally occurring ribozymes [1].

Group I introns vary considerably in complexity. Most

introns contain only the sequence information required for

splicing, whereas others contain large extensions of the

peripheral domains. Some of the larger group I introns

contain an open reading frame, usually represented by a

homing endonuclease gene (HEG). HEGs are found in

different conﬁgurations, e.g. fused in frame with the

upstream exon or as an independent expression unit [2].

The most complex group I introns are the twin-ribozyme

introns that in addition contain a group I-like cleavage

ribozyme (GIR1) involved in the expression of the intron

HEG [3].

The complex structure of the twin-ribozyme introns

suggests a complex biology. This has been demonstrated in

the case of the Dir.S956-1 (former DiSSU1; the recently

introduced nomenclature for group I introns [4] is used

throughout this paper) intron found in the small subunit

ribosomal RNA (SSU rRNA) gene in the myxomycete

Didymium iridis (Fig. 1). One of the ribozymes (DiGIR2)

catalyses intron excision and exon ligation (Fig. 1, left

panel). In addition, this ribozyme displays a pronounced 3¢

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

question of its formation and possible function in cellular

I-DirI expression, we here investigate its identity and

distribution. We demonstrate that the RNA is a 7.5-kb

Correspondence to H. Nielsen, Department of Biochemistry and

Genetics, Laboratory B, The Panum Institute, Blegdamsvej 3,

DK-2200 N, Denmark. Fax: +45 35 32 77 32, Tel.: +45 35 32 77 63,

E-mail: hamra@imbg.ku.dk

Abbreviations:DiGIR,Didymium group I ribozyme; SSU, small

subunit; LSU, large subunit; ETS, external transcribed spacer; ITS,

internal transcribed spacer; HEG, homing endonuclease gene;

IPS, internal processing site.

(Received 24 July 2002, revised 24 September 2002,

accepted 30 September 2002)

Eur. J. Biochem. 269, 5804–5812 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03283.x

linear species generated by an unusual pre-rRNA processing

event mediated by DiGIR1, and that it accumulates during

starvation-induced encystment in D. iridis.

EXPERIMENTAL PROCEDURES

Cell cultivation, RNA isolation and Northern blotting

analysis

The intron-containing D. iridis strain Lat3-5, derived from

the Pan2-44 isolate, has been described previously [8]. The

cells were cultured at 26 C in liquid media (DS/2;

1mgÆmL

-glucose, 0.5 mgÆmL

yeast extract,

0.1 mgÆmL

MgSO

,1mgÆmL

,1.5mgÆmL

HPO

) 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

For RNA extraction, a total of 10

Didymium cells

were harvested by centrifugation at 400 gfor 5 min The

pellet was dissolved in 1 mL Trizol Reagent (Gibco-BRL)

and RNA extracted according to the manufacturer’s

instructions. Aliqouts of RNA were denatured for 15 min

at 65 C in loading buffer (1 ·Mops, 17.8% formalde-

hyde, 50% formamide, 12 ngÆlL

ethidium bromide) and

fractionated on a 5.3% formaldehyde/1% agarose gel in

5.3% formaldehyde/1 ·Mops (40 m

Mops, 10 m

NaAc, 2 m

EDTA, 0.04% HAc). The RNA was then

transferred to a nylon membrane by Northern blotting

using capillary action. Hybridization was carried out

either in Rapid Hyb solution (AP Biotech) or in Ultrahyb

solution (Ambion) according to the manufacturer’s

instructions.

The external transcribed spacer (ETS), GIR1, HEG,

GIR2, internal transcribed spacer (ITS)1, ITS2 and large

subunit (LSU)1 probes were ampliﬁed from Lat3-5 genomic

DNA [8] by PCR using the oligo pairs OP448/OP449,

OP20/C78, OP1/OP2, OP11/OP180, SSU6/SSU7, C231/

C232 and OP65/OP169, respectively. The sequences of the

oligonucleotides are: OP1, 5¢-CACTTCTAGAACCA

TGGTGAAAGGAACG-3¢;OP2,5¢-TGTCTGGATCCT

CATCTG-3¢;OP4,5¢-TGTTGAAGTGCACAGATT-3¢;

OP11, 5¢-GACTAGTTGACTTCTCACAGA-3¢; OP20,

5¢-TTGAACACTTAATTGGGT-3¢;OP65,5¢-GGAG

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.

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-

P]dCTP (3000 CiÆmmol

;

Amersham). The ITS1 and ITS2 PCR products were

cloned using the Topo TA cloning kit (version J, Invitrogen)

according to the manufacturer’s instruction. Plasmids

harbouring the ITS1 insert in the correct orientation were

linearized by HindIII digestion, while the ITS2 insert was

further subcloned into the XbaI/HindIII site of the pBlue-

script+ vector (Stratagene) to obtain the correct orienta-

tion. The resulting pBluescript plasmid was linearized with

XbaI.

Riboprobes were transcribed from 500 ng linearized

template DNA using 500 l

each of rATP, rCTP and

rGTP, 25 l

rUTP, 0.5 l

[a-

P]UTP (3000 CiÆmmol

;

Amersham), 10 m

dithiothreitol and 50 U T7 RNA

polymerase (Stratagene) in 20 lLof1·the supplied buffer

at 37 Cfor1h.

RNaseH analysis

A mix consisting of 6 lg RNA and 50 pmol oligo was

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-

P]ATP (3000 CiÆmmol

, Amersham) using T4 poly-

nucleotide kinase (Gibco-BRL). RNA was added to 2 pmol

labelled oligo in 1 ·RT buffer (50 m

Tris/HCl at pH 8,

60 m

KCl, 10 m

MgCl

,1m

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

(Promega), 0.2 m

dATP, dCTP and dTTP and 0.4 m

dGTP (Pharmacia) in 1 ·RT buffer. The reaction was

incubated1 hat40 C before being stopped by the addition

of 5 lL formamide loading buffer. The primer extension

product was denatured by heating at 100 Cfor1min

before separation on an 8

urea/8% polyacrylamide gel.

Cell fractionation and sucrose gradients

DS/2 (see above) was added to 2 ·10

Lat3-5 cells to a total

volume of 2 ·14 mL and centrifuged in two tubes at 300 g

for 5 min The pellet from one tube was dissolved in 1 mL

Trizol (see above) for extraction of total RNA. The cells in

the other tube were resuspended in 250 lL ice-cold lysis

buffer (20 m

Tris/HCl pH 8.0, 1.5 m

MgCl

,140m

KCl, 1.5 m

dithiothreitol, 1 m

CaCl

,0.1m

EDTA,

0.16 m

cycloheximide, 0.5% Nonidet P-40, 500 UÆmL

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

Tris/HCl at

pH 7.5, 100 m

LiCl, 10 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.

RESULTS

A 7.5-kb I-DirI HEG RNA signal is enriched upon

starvation of Didymium cells

The life-cycle of a typical myxomycete can be roughly

divided into a diploid macroscopic stage consisting of a

plasmodium and the fruiting bodies that develop from it,

and a haploid microscopic stage (see Fig. 1B). Haploid

myxomycete cells are uninucleate and exist in two intercon-

vertible active states; nonpolarized amoebae and polarized

ﬂagellated swarm cells. The particular form in which a given

cell exists depends largely upon the amount of water in the

environment, with swarm cells tending to dominate under

aqueous conditions. In nature, myxamoebae or swarm cells

feed by phagocytosis of bacteria. Under conditions unfa-

vourable for continued growth, such as starvation, the

vegetative cells will develop into dormant cysts. Cysts can

remain viable for long periods of time, and have been

suggested to be very important for the survival of

myxomycetes in some habitats.

To examine whether the cellular amount of the 7.5-kb

I-DirI HEG RNA correlates with food availability, intron-

harbouring Lat3-5 amoebae were grown in monoxenic

culture using E. coli as a food source (Fig. 2A). As the

cells grow and multiply, food is depleted (time points 1–5)

and the myxamoebae gradually transform into very active

swarm cells (points 5–6). Eventually, activity ceases and the

starving cells develop into dormant cysts (point 7). As

encystment is deﬁned by the formation of a cell wall, we

have chosen to score cysts by their ability to resist lysis in

0.5% Nonidet P40 [11]. However, it is important to keep in

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)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

know )from repeated experiments using in vitro tran-

scribed and processed RNA )that circles are retarded and

thus efﬁciently separated from the corresponding linear

form of the intron RNA. Northern blotting analysis showed

that an RNA with the same migration as a Dir.S956-1 circle

is indeed present in Didymium cells, but that this RNA is

enriched at the start of the time course rather than during

starvation (data not shown). Second, the circular and linear

forms of Dir.S956-1 RNA from an in vitro splicing reaction

were separated on a denaturing polyacrylamide gel, cut out

and recovered after elution. The RNA species were analysed

on a denaturing agarose gel. The results showed that the

Dir.S956-1 circle is only slightly retarded on an agarose

gel (data not shown). Thus, contrary to our previous

Fig. 2. Analysis of RNA from D. iridis Lat3-5 cells harvested from a time course growth experiemt. (A) Time course of culture growth, showing

starvationandsubsequentencystmentofvegetativeD. iridis Lat 3-5 cells. The time points when total number of Didymium cells (d), number of

encysted cells (s)oramountofE. coli food (j) was measured are indicated. The numbered arrows denote the time points when RNA samples were

obtained. (B) Northern blot of Lat3-5 whole cell RNA. Didymium cells (5 ·10

) were harvested at the time points indicated in (A). After extraction,

the RNA was separated on a 1% denaturing agarose gel and analysed by Northern blotting analysis using the HEG probe described in Fig. 3A. An

overview of the observed intron RNA species is shown to the right. Exon, open reading frame and intron sequences are indicated in black, grey and

white, respectively. The position of the 5¢and 3¢splice sites (SS) as well as internal processing sites (IPS) are indicated. The 1.46-kb RNA is the full-

length excised Dir.S956-1 intron, while 1.23-kb RNA and 0.85-kb RNA represent processed forms of the intron RNA [7]. The 7.5-kb signal under

investigation is denoted**, while the identity of the signal marked * is discussed in the text. The size indications are derived from the 0.24–9.5 kb

ladder (GibcoBRL) visualized by ethidium bromide staining.

FEBS 2002 Ribozyme mediated processing of pre-rRNA (Eur. J. Biochem. 269) 5807

suggestion, the 7.5-kb signal does not correspond to the full

length circular intron RNA.

Next, we hypothesized that the 7.5-kb RNA is formed by

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

SSU and LSU rRNA genes, as well as the position of the Dir.S956-1, Dir.L1949 and Dir.L2449 group I introns [9,18]. Exon and intron sequences

are denoted in black and white, respectively. The localization of the probes applied for Northern blotting analyses is indicated with thick black lines.