The group I-like ribozyme DiGIR1 mediates alternative processingAnna Vader1,2, Steinar Johansen2and Henrik Nielsen1 1 Department of Medical Biochemistry and Genetics, The Panum Institute
Trang 1The group I-like ribozyme DiGIR1 mediates alternative processing
Anna Vader1,2, Steinar Johansen2and Henrik Nielsen1
1
Department of Medical Biochemistry and Genetics, The Panum Institute, Copenhagen, Denmark;2Department 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 RNAthat 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 RNAretains 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 rRNAlacking its two
group I introns The formation of this RNAimplies
clea-vage by DiGIR1 in a new RNAcontext, and presents a new
example of the cost to the host of intron load This is because
the formation of the 7.5-kb RNAis incompatible with the formation of functional ribosomal RNAfrom 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 rRNAand I-DirI mRNAin growing cells, suggesting an interplay between the two ribozymes of a twin-ribozyme intron
Keywords: Didymium iridis; group I intron; ribozyme; pre-rRNAprocessing
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 RNAcircles 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-DirI mRNA and is 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 RNAis removed before the resulting I-DirI mRNAis 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 Didymiumisolates [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 RNArepresented 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 iridisculture when the RNAwas 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 RNAis 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
Trang 2linear species generated by an unusual pre-rRNAprocessing
event mediated by DiGIR1, and that it accumulates during
starvation-induced encystment in D iridis
E X P E R I M E N T A L P R O C E D U R E S
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 26C in liquid media (DS/2;
1 mgÆmL)1 D-glucose, 0.5 mgÆmL)1 yeast extract,
0.1 mgÆmL)1MgSO4, 1 mgÆmL)1KH2PO4, 1.5 mgÆmL)1
K2HPO4) 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/Pi
For RNAextraction, a total of 107 Didymium cells
were harvested by centrifugation at 400 g for 5 min The
pellet was dissolved in 1 mL Trizol Reagent (Gibco-BRL)
and RNAextracted according to the manufacturer’s instructions Aliqouts of RNA were denatured for 15 min
at 65C in loading buffer (1 · Mops, 17.8% formalde-hyde, 50% formamide, 12 ngÆlL)1ethidium bromide) and fractionated on a 5.3% formaldehyde/1% agarose gel in 5.3% formaldehyde/1· Mops (40 mM Mops, 10 mM NaAc, 2 mM 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 RNAspecies 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-rRNAand 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 RNAis formed after the excision of the LSU rRNAintrons Dir.L1949 and Dir.L2449 The accumulated 7.5-kb RNAcontains all of the Dir.S956-1 sequences except those encoding the cleavage ribozyme DiGIR1 Apossible functional role of the 7.5-kb RNAis as an alternative precursor for the endonuclease mRNAduring excystment Here, the HEG RNAmight be separated from the remaining 7.5-kb RNAsequences 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 RNAalso 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.
Trang 3GTTCAGAGACTATA-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-32P]dCTP (3000 CiÆmmol)1;
Amersham) The ITS1 and ITS2 PCR products were
cloned using the Topo TAcloning 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 DNAusing 500 lM each of rATP, rCTP and
rGTP, 25 lMrUTP, 0.5 lM[a-32P]UTP (3000 CiÆmmol)1;
Amersham), 10 mM dithiothreitol and 50 U T7 RNA
polymerase (Stratagene) in 20 lL of 1· the supplied buffer
at 37C for 1 h
RNaseH analysis
Amix consisting of 6 lg RNAand 50 pmol oligo was
heated in 1· RNaseH buffer (GibcoBRL) at 80 C for
1 min A t 45C, 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 30C for
5 min, prior to analysis by Northern blotting (see above)
Primer extension
For primer extension, gel-purified OP4 was labelled with
[a-32P]ATP (3000 CiÆmmol)1, Amersham) using T4
poly-nucleotide kinase (Gibco-BRL) RNAwas added to 2 pmol
labelled oligo in 1· RT buffer (50 mMTris/HCl at pH 8,
60 mMKCl, 10 mMMgCl2, 1 mMdithiothreitol) in a total
volume of 5 lL, denatured at 80C for 2 min and
incubated at 45C for 10 min Subsequently 4 lL RNA /
oligo mixture was added to a tube containing 1 U AMV
reverse transcriptase (RT; Pharmacia), 1 U RNasin
(Promega), 0.2 mM dATP, dCTP and dTTP and 0.4 mM
dGTP (Pharmacia) in 1· RT buffer The reaction was
incubated 1 h at 40C before being stopped by the addition
of 5 lL formamide loading buffer The primer extension
product was denatured by heating at 100C for 1 min
before separation on an 8Murea/8% polyacrylamide gel
Cell fractionation and sucrose gradients
DS/2 (see above) was added to 2· 107Lat3-5 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 mM Tris/HCl pH 8.0, 1.5 mM MgCl2, 140 mM KCl, 1.5 mM dithiothreitol, 1 mMCaCl2, 0.1 mMEDTA, 0.16 mM 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, 4C 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 RNAwas heated to 70C for 5 min, cooled on ice and centrifuged at
13 000 g, 4C for 5 min The supernatant was loaded onto
a linear 15–40% sucrose gradient in 10 mM Tris/HCl at
pH 7.5, 100 mMLiCl, 10 mMEDTAand 0.2% SDS and centrifuged for 20 h at 4C and 25 000 r.p.m in a Beckman SW27.1 rotor Fractions of approximately 1 mL were collected and RNAwas isolated by phenol/chloroform extraction
R E S U L T S
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 RNAcorrelates 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 RNAfrom a time course of a Didymium culture shows that the 7.5-kb RNAis 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 RNAin the cells While the amounts of some of the other HEG RNAspecies 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 increadecrea-ses The 3.9 kb RNAappears
5806 A Vader et al (Eur J Biochem 269) FEBS 2002
Trang 4to be a nuclear species [7], and a similar RNAhas been
observed when whole cell RNAfrom 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 RNAis independent of the intron, and results
from a more general alternative pathway of Didymium
pre-rRNAprocessing
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, RNAfrom 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 RNAwith the same migration as a Dir.S956-1 circle
is indeed present in Didymium cells, but that this RNAis 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 RNAfrom an in vitro splicing reaction were separated on a denaturing polyacrylamide gel, cut out and recovered after elution The RNAspecies 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 Lat3-5cells harvested from a time course growth experiemt (A) Time course of culture growth, showing starvation and subsequent encystment of vegetative D iridis Lat 3-5 cells The time points when total number of Didymium cells (d), number of encysted cells (s) or amount of E coli food (j) was measured are indicated The numbered arrows denote the time points when RNAsamples were obtained (B) Northern blot of Lat3-5 whole cell RNA Didymium cells (5 · 10 6 ) were harvested at the time points indicated in (A) After extraction, the RNAwas 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 RNAspecies 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 RNAis the full-length excised Dir.S956-1 intron, while 1.23-kb RNAand 0.85-kb RNArepresent 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.
Trang 5suggestion, the 7.5-kb signal does not correspond to the full
length circular intron RNA
Next, we hypothesized that the 7.5-kb RNAis formed by
an alternative processing of the pre-rRNAin which the SSU
rRNAsequence 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 RNAcompared with ribosomal
RNA species, and the fact that it coexists with RNAs
containing the same structural elements, we decided to
deduce its structure by analysis of preparations of whole cell
RNArather than to isolate it Whole cell RNAwas isolated
from Didymium cells harvested early and late in a time
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-rRNAincluding 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-rRNAsubsequent to Dir.S956-1 excision The observation of the 9.5 kb RNAis 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-rRNAis not available, the size of 9.5 kb for this RNAis
in reasonable agreement with the expected size based
on reported ribosomal DNAsequences from different Didymiumisolates
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 rRNAgenes, 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 The lower panel is an enlarged segment of the upper panel, and shows the positions of the oligonucleotides used in RNaseH analysis (B) Northern blotting analysis of whole cell RNA Parallel filters containing RNA from Didymium cells harvested at an early (E) and late (L) time point (corresponding to positions 1 and 6 in Fig 2A) were hybridized with the probes indicated The size indications are derived from the 0.24- to 9.5-kb ladder (GibcoBRL) visualized by ethidium bromide staining (C) RNaseH analysis of the 7.5-kb RNAsignal Whole cell RNAfrom a late time point (corresponding to point 6 in Fig 2A) was fractionated on a 15–30% sucrose gradient (see Fig 5) Fractions enriched in the 7.5-kb RNA, as determined by Northern blotting analysis, were pooled and the extracted RNAsubjected to RNaseH analysis with the oligonucleotides indicated The resulting RNAwas analysed by Northern blotting using the HEG probe shown in (A) The size indications are derived from the High Range RNAladder (Fermentas) visualized by ethidium bromide staining.
5808 A Vader et al (Eur J Biochem 269) FEBS 2002
Trang 6The RNAspecies that are involved in expression of the
HEG, and particularly those that are involved in the
formation of the 7.5-kb RNA, are considerably less
abundant than most of the ribosomal RNAprecursors
For this reason, detection of HEG RNAs with non-HEG
probes was technically difficult Nevertheless, a 7.5-kb signal
specific for late RNAwas identified by the ITS1 and ITS2
probes, but was absent when a 5¢ ETS probe was used
(Fig 3B) The 7.5-kb RNAwas furthermore detected by
exonic LSU probes but was not detected by probes targeted
towards the LSU introns (data not shown) The absense of
the LSU introns in the 7.5-kb RNAimplies a different
splicing order during the formation of this RNAcompared
with normal processing of the pre-rRNAin which the
Dir.S956-1 sequences are removed prior to the LSU introns
(see above) The expected size of an RNAcomposed of the
elements suggested by the Northern blotting experiment is
7.6 kb which is in good agreement with the observed size
of 7.5 kb
In the RNaseH analysis, the parallel RNAsamples
were hybridized to oligonucleotides complementary to
different parts of the Didymium pre-rRNA(see Fig 3A)
RNaseH, which will degrade the RNAstrand of the
resulting RNA: DNA heteroduplexes, was then added
The resulting RNAfragments 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 RNAand 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 RNAsignal 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
hybridize to different parts of the HEG or GIR2 led to
cleavage (data not shown) The sizes of the upstream
cleavage products, detected by their hybridization to a HEG
probe in Northern blotting analysis, suggested that the 5¢
end of the 7.5-kb RNAwas located at or near the internal
processing sites IPS1/2
RNaseH analysis was also used to examine whether the
51-nucleotide spliceosomal intron within the HEG is present
in the 7.5-kb RNA This intron is removed during
maturation of the I-DirI mRNA from the excised
Dir.S956-1 RNA[7] Cleavage with an oligonucleotide
complementary to the spliceosomal intron sequences
showed that these are retained in the 7.5-kb RNA(data not shown)
The DiGIR1 ribozyme catalyses 5¢ end formation of the linear 7.5-kb RNA
To map precisely the 5¢ end of the 7.5-kb RNA, it was necessary to separate it from other Dir.S956-1-containing RNAs in the cell Whole cell RNA from a late time point was fractionated by ultracentrifugation through a 15–40% sucrose gradient (Fig 5A) Screening of the collected fractions by Northern blotting analysis, showed that the HEG-containing RNAs could successfully be separated according to size (Fig 5B) Primer extension with an oligo complementary to the 5¢ end of the HEG produced only one signal which corresponded to IPS2 (Fig 5C) The intensity of the signal through the fractions suggested that two populations of HEG RNAs with this end exist in the
Fig 4 Characterization of the intronic part of the linear 7.5-kb RNA (A) Schematic presentation of the Dir.S956-1 intron, showing the 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 RNAfrom 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 RNAmarker (Promega) was used as a size marker.
Trang 7cells 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 RNAand
0.85-kb RNA; see Fig 2) are located Another
IPS2-termin-ated HEG RNAexists in the high molecular fractions
where the 7.5-kb RNAis 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 RNAhas been
detected in vivo [7] We infer that 5¢ end formation of the
7.5-kb RNAis a result of DiGIR1 catalysis The critical
involvement of DiGIR1 in the formation of the 5¢ end of
the RNAcould 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-DirI HEG RNAs differ in their intracellular distribution While the fully processed 0.85-kb RNAis located almost exclusively in the cytosol, all other examined HEG RNAs (3.9 kb, 1.46 kb, and 1.23 kb RNA, respectively; see Fig 2) are nuclear [7] In order to examine the subcellular location of the 7.5-kb RNA, Didymium Lat3-5 cells from a late time point (corresponding to 6 in Fig 2A) were lysed and fractionated by centrifugation and RNAisolated from the nuclear and cytosolic fractions Although we had some difficulties recovering large RNAs after the fractionation procedure, probably due to high nuclease activity in the encysting cells, the resulting Northern blot indicated a nuclear localization for the 7.5-kb RNA(Fig 6) As expected, the 0.85-kb RNAwas the only HEG RNA species to be found in the cytosol As such, it provided
an internal control for the success of the fractionation procedure
D I S C U S S I O N
We have previously noted the presence of an I-DirI HEG RNAsignal corresponding to 7.5 kb when D iridis Lat3-5 RNAwas analysed by Northern blotting [7] We show here that this RNAis a linear species, produced by alternative processing of the pre-rRNA The 5¢ end of the RNA corresponds to an internal processing site in Dir.S956-1 (IPS2), implying that it is formed through cleavage by the DiGIR1 ribozyme The 3¢ end has not been mapped but is assumed to correspond approximately to the 3¢ end of the pre-rRNA Apart from two group I introns in the LSU rRNA(Dir.L1949 and Dir.L2449), the 7.5-kb RNA
Fig 6 Intracellular localization of the 7.5-kb RNA Total (T), nuclear (N) and cytosolic (C) RNAfrom 5 · 10 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 RNAladder (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 RNAand 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
was also used to make a DNA sequencing ladder which was used to
determine the exact position of the primer extension stop at IPS2 as
indicated.
5810 A Vader et al (Eur J Biochem 269) FEBS 2002
Trang 8contains all sequence elements downstream of IPS2 (see
Fig 1, right panel) These include the LSU rRNAexons,
the internal transcribed spacers (ITS1 and ITS2) and the
part of the SSU rRNAthat is located downstream of
Dir.S956-1 All intron sequences downstream of IPS2,
including the spliceosomal intron within the I-DirI protein
coding region, are also present The RNAis localized in the
nucleus, and accumulates when Didymium cells are starved
In cells about to form dormant cysts, the 7.5-kb RNAis the
predominant I-DirI HEG RNA When viewed as a
nonribosomal RNA, it is relatively abundant and
remark-ably stable considering the turn-over of other RNAs,
including ribosomal RNAs, that take place during
encystment
The existence of the 7.5-kb I-DirI HEG RNA is
surprising, as the molecule contains several intrinsic
activities and signals that would be expected to result in
its disappearance Firstly, even though the cleavage by
DiGIR1 at IPS2 removes the 5¢ splice site site of
Dir.S956-1, the catalytic core of the DiGIR2 splicing
ribozyme remains intact DiGIR2 RNAs with even larger
truncations in the 5¢ end have previously been shown to
perform efficient hydrolysis at the 3¢ splice site of
Dir.S956-1 [5] Secondly, an internal processing site
(IPS3) is located downstream of the HEG region The
site is cleaved during maturation of the mRNAfrom the
excised intron [7] Thirdly, the 7.5-kb RNAcontains a
polyadenylation signal, which in mRNAformation
indu-ces cleavage and polyadenylation of the I-DirI mRNA
Finally, a spliceosomal intron (I51) harboured by the
I-DirI HEG is removed in the mature I-DirI mRNA[7]
All of the activities mentioned above would be expected
to act against the preservation of the 7.5-kb RNA
The composition of the 7.5-kb RNAsuggests 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-rRNAand pre-mRNAslows down
when cells are starved In the ciliate Tetrahymena
pyrifor-mis, the pre-rRNAprocessing 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 RNAis expected as the RNA
retains several putative nuclear retention signals, e.g a
spliceosomal intron
The accumulation of the 7.5-kb RNAduring
starva-tion does not necessarily imply that this RNAis specific
for starved cells It is possible that the 7.5-kb RNAis a
default intermediate in the formation of the I-DirI
mRNAthat 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
exclude this possibility but consider it unlikely in view of the high and comparable levels of the excised intron and precursors for the I-DirI mRNAfound in exponentially growing cells [7] In any case, the accumulation of the 7.5-kb RNAimplies an alternative processing pathway for pre-rRNA
Interestingly, the relative activities of the splicing and cleavage ribozymes seem to be altered when the formation
of the 7.5-kb RNAis compared to the formation of other HEG RNAs In the formation of mature rRNA from pre-rRNA, excision of the optional Dir.S956-1 intron precedes that of the two conserved LSU introns Furthermore, DiGIR1 seems to be active only after splicing by DiGIR2 has taken place [5,7,9] In the 7.5-kb RNA, on the other hand, Dir.L1949 and Dir.L2449 have been removed, while the catalytic part of DiGIR2 is still present and DiGIR1 is active without prior splicing In addition, we have recently discovered that full-length circles are formed in a reaction pathway catalysed by DiGIR2 and that DiGIR1 is com-pletely inactive in this pathway (unpublished data) These observations suggest that some mechanism is in operation
to modulate the activities of DiGIR1 and DiGIR2 with respect to each other leading to different processing products
What can be the function of the 7.5-kb RNA? During cyst formation, cells actively degrade intracellular sub-stances In a study of the myxomycete Physarum flavico-mum it was found that the content of protein, neutral hexose and RNAdecreased by 40, 41 and 21%, respect-ively [15] At the same time, excystment has been reported
to occur even in the presence of 300 lgÆmL)1 actinomy-cin D, suggesting that new RNAsynthesis 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 RNAis a dead-end product, it remains a possibility that it functions as a precursor for the I-DirI mRNAduring excystment Preliminary experi-ments aimed at following the 7.5-kb RNAthrough germination of cysts have failed due to lack of synchrony
in cultures of germinating cysts
In conclusion, we have described a new RNAspecies that accumulates by alternative processing of pre-rRNAduring starvation-induced encystment in Didymium The most interesting aspect of this RNAis 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 RNAis incompatible with the formation of ribosomal RNAfrom the same transcript It shows DiGIR1 activity in
a different RNAcontext 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
A C K N O W L E D G E M E N T S
We thank F Frenzel for technical assistance and J Christiansen for helpful suggestions to the experiments This work was supported by grants from the Norwegian Research Council (AV), the Danish Research Council for Natural Sciences (HN), and The NOVO Foundation (HN).
Trang 9R E F E R E N C E S
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