From the Institutes of Biopharmaceutical
Science and § Biochemistry, National Yang-Ming University,
Taipei 112, Taiwan, Republic of China
Received for publication, August 15, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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In Saccharomyces cerevisiae, Cdc13p
is a single-stranded TG1-3 DNA binding protein that
protects telomeres and maintains telomere length. A mutant allele
of CDC13, cdc13-1, causes accumulation of
single-stranded TG1-3 DNA near telomeres along with a G2/M cell cycle arrest at non-permissive temperatures. We
report here that when the single-stranded TG1-3 DNA is
masked by its binding proteins, such as S. cerevisiae Gbp2p
or Schizosaccharomyces pombe Tcg1, the growth arrest
phenotype of cdc13-1 is rescued. Mutations on Gbp2p that
disrupt its binding to the single-stranded TG1-3 DNA
render the protein unable to complement the defects of
cdc13-1. These results indicate that the presence of a
single-stranded TG1-3 tail in cdc13-1 cells
serves as the signal for the cell cycle checkpoint. Moreover, the
binding activity of Gbp2p to single-stranded TG1-3 DNA
appears to be associated with its ability to restore the
telomere-lengthening phenotype in cdc13-1 cells. These
results indicate that Gbp2p is involved in modulating telomere length.
Telomeres are the structure at the ends of eukaryotic
linear chromosomes (1, 2). In most organisms, the telomeric DNA is
composed of short, tandem repeated sequences with a strand rich in
guanine residues (G-strand) running 5' to 3' toward the end of
telomere. For example, the telomeric sequences in the brewers yeast
Saccharomyces cerevisiae are ~250-300 base pair-long
TG1-3/C1-3A repeats. Sequences of ciliate
telomeres reveal that the G-strand extends beyond the duplex region,
creating a short single-stranded 3'-overhang (3-5). In yeast, longer
G-strand DNA with varying lengths, presumably an intermediate during
telomere replication, is detected during late S phase (6, 7).
Telomeres are essential for the maintenance of chromosome
integrity. Telomeres protect chromosomes from degradation by nucleases,
facilitate complete replication of chromosomes, and differentiate
linear chromosome ends from broken ends (1, 2).
Several single-stranded telomeric DNA binding proteins, including
Oxytricha A mutation allele of CDC13, cdc13-1, causes
yeast cells to arrest at the G2/M phase of the cell cycle
and accumulate single-stranded TG1-3 DNA at telomeres
(20). Various types of DNA damages, including x-ray, UV, and chemical
mutagens, induce DNA damage-dependent cell cycle arrest
(24-28). Presumably, these damages on DNA were processed by cellular
nucleases to generate single-stranded DNA, which could then serve as
the signal for DNA damage-dependent cell cycle arrest (20,
29). Here, we show that S. cerevisiae Gbp2p and S. pombe Tcg1 rescue the growth arrest of cdc13-1 cells and that their single-stranded TG1-3 DNA binding activity is required for their complementation. Our results suggest that the
appearance of single-stranded telomeric DNA in cdc13-1
cells is the signal that induces the DNA damage-dependent
checkpoint. Moreover, we also provide evidence that Gbp2p is involved
in modulating telomere length.
Yeast Strains--
Yeast strain W13a (MATa cdc13-1 his7
leu2-3, 112 ura3-52 trp1-289) and the wild-type CDC13
version of W13a, strain 4053-5-2a, were used as the hosts in
complementation tests (provided by L. Hartwell, Fred Hutchinson Cancer
Research Center). Strain YEM1 Plasmids--
Plasmids pTHA, pTHA-CDC13 (expresses
CDC13 under the control of PGK1 promoter; Ref.
11) and pGAL-GBP2 (expresses GBP2 under the control of
GAL1 promoter; Ref. 12) were described previously. To
express S. cerevisiae HRB1 in S. cerevisiae, HRB1 was first PCR amplified with primers
HRB15 (5'-CCCATGGGTGATGATCATGGTTAT-3') and HRB13
(5'-AGGCCTAGAGGCGTTTAGCGATCG-3') using Vent DNA polymerase (New
England Biolabs). The ~1.1-kbp HRB1 PCR fragment was
cloned directly into pGEM-T (Promega) to generate pGEM-HRB1. To
construct GAL1 promoter-driven HRB1, the
NcoI-StuI HRB1 DNA-containing fragment from pGEM-HRB1 was ligated into the NcoI- and
SmaI-digested
pSH380.1
DNA fragments used in two-hybrid analysis were subcloned into pEG202
(Ampr HIS3 2µ) or pJG4-5
(Ampr TRP1 2µ) (31). To construct
pJG-GBP2, the GBP2 fragment was first PCR amplified with primers GBP25
(5'-GCGAATTCACCATGGAGAGAGAGCTA-3') and GBP23
(5'-GCCTCGAGGTTGGTGCTTAGGAATTA-3').
Cloning and Sequencing of S. pombe TCG1--
To clone S. pombe genes that complement the cdc13-1
temperature-sensitive phenotype, an S. pombe cDNA
library (32) was transformed into W13a. Transformants were plated,
incubated at 25 °C for 16 h, and then at 30 °C for 4 days. A
total of 72 colonies grew at 30 °C from ~120,000 transformants.
DNAs from these 72 colonies were prepared and transformed into
Escherichia coli XL1-Blue. Plasmid DNAs were prepared and
transformed back into W13a to retest their temperature sensitivity at
30 °C. Only one clone grew at 30 °C after the retransformation of
purified plasmids. A 1.4-kbp HindIII-HindIII DNA
insert within this complementing plasmid, pDB-TCG1, was subcloned into
HindIII-digested pVZ1 to generate pVZ-TCG1. The sequence of
this DNA was determined by the exonuclease III deletion method and
sequenced using Sequenase (Upstate Biotechnology). To construct
pGST-TCG1, the TCG1 fragment was first PCR amplified with
primers TCG5 (5'-CGAATTCATGATGTCCGCTGAGGAAAC-3') and TCG3 (5'-CGTCGACTTAGGCAGTTATAGCACTAG-3'). A PCR-amplified ~1.0-kbp DNA
fragment was digested with EcoRI and SalI and
ligated with the EcoRI- and XhoI-digested
pGEX-4T-1 to generate pGST-TCG1.
Site-specific Mutagenesis--
PCR-based site-specific
mutagenesis was applied to generate mutations on the RNA recognition
motifs (RRMs) of GBP2. Mutations on residues Arg-161
and Arg-259 of Gbp2p were changed to Ala on plasmid pMAL-GBP2 to
generate pMAL-GBP2R161A and pMAL-GBP2R259A,
respectively. The mutation pMAL-GBP2R161A, R259A was
generated by conducting a second mutagenesis on plasmid
pMAL-GBP2R259A by changing Arg-161 to Ala. A similar
approach was applied to generate mutations on
pGAL-GBP2R161A, pGAL-GBP2R259A, and
pGAL-GBP2R161A, R259A. All the mutations were confirmed by
DNA sequencing.
Protein Purification--
To purify glutathione
S-transferase
(GST)2-fused Tcg1, a 1000-ml
culture of E. coli harboring pGST-TCG1 was grown at 30 °C to an A600 of 0.8 and induced with the
addition of 1 mM IPTG. The cells were grown at 30 °C for
another 3 h before being harvested by centrifugation. Cells were
resuspended in 20 ml of sonication buffer (50 mM Tris-HCl,
pH 8.0, 1 mM EDTA, 0.5% Triton X-100, 1 mM
DTT, 50 mM NaOAc, 20% glycerol, and 1× protease
inhibitors (Calbiochem)) and sonicated to release the cell contents.
The sonicated cells were centrifuged at 13,000 × g for
15 min at 4 °C to get rid of soluble cell fractions. The pellet was
then extracted with sonication buffer with 0.25% Tween 20 and 0.1 mM EGTA to obtain GST-Tcg1 fusion protein. Purified protein
was dialyzed against storage buffer (50 mM Tris-HCl, pH
8.0, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT,
50 mM NaOAc, 50% glycerol), aliquoted, and frozen by dry
ice-ethanol bath.
To purify maltose-binding protein (MBP)-fused Gbp2p, 100 ml culture of
E. coli harboring pMBP-GBP2, was grown at 30 °C to an
A600 of 0.5 and induced with the addition of 1 mM IPTG. The cells were grown at 30 °C for another
16 h before being harvested by centrifugation. Cells were
resuspended in 10 ml of sonication buffer (50 mM
NaH2PO4, pH 7.8, 1 mM EDTA, 0.5%
Triton X-100, 1 mM DTT, 5% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 1× protease inhibitors
(Calbiochem)) and sonicated to release the cell contents. The sonicated
cells were centrifuged at 13,000 × g for 15 min at
4 °C to obtain total cell-free extracts. 0.5 ml of amylose-agarose
(New England Biolabs) was added to the total cell-free extracts and
incubated at 4 °C for 1 h. The resin was washed and eluted with
2 ml of buffer containing 50 mM
NaH2PO4, pH 8.0, 100 mM maltose,
and 20% glycerol. Purified protein was aliquoted and frozen by a dry
ice-ethanol bath. Gbp2p with different point mutations were purified
using similar procedures.
Electrophoretic Mobility Shift Assay (EMSA)--
Oligonucleotide
TG43 (5'-GTGGTGGGTGGGTGTGTGTGGGTGTGGTGGGTGTGTGGGTGTG-3') was
labeled with [ Single-stranded Telomere Tail and Telomere Length
Determinations--
Yeast cells CDC13/pSH380 or cdc13-1
cells harboring plasmid pSH380, pGAL-GBP2, pGAL-GBP2R161A,
pGAL-GBP2R259A, or pGAL-GBP2R161A, R259A were
grown in YC-Leu in the presence of 2% glucose at 25 °C for 2 days.
Cells were then washed with water, resuspended in YC-Leu medium with
3% galactose, and continued to grow at 30 °C for another 6 h.
DNA from these cells was prepared, digested with XhoI, and separated on 1% agarose gels. Single-stranded telomere tails were determined by nondenaturing Southern hybridization as described previously (6). To determine the telomere length, DNA fragments were
transferred to a Hybond N+ paper (Amersham Pharmacia
Biotech) for hybridization using a random primed
C1-3A/TG1-3 DNA probe (12).
In S. cerevisiae, a cdc13-1
mutation causes yeast cells to arrest at the G2/M phase and
accumulate single-stranded TG1-3 DNA at telomeres at
non-permissive temperatures (20). Analogous to what is caused by
various types of DNA damage, the accumulated single-stranded telomeric
DNA may serve as the signal for cell cycle arrest in
cdc13-1 cells (20, 29). In this scenario, it is
conceivable that single-stranded telomeric binding proteins in excess
amount could mask the single-stranded DNA present in cdc13-1 cells at the non-permissive temperature and prevent
the G2/M arrest. To test this possibility, genes encoding
single-stranded TG1-3 DNA binding proteins were expressed
in cdc13-1 cells to see if they rescue the
temperature-sensitive phenotype (Fig. 1).
It has been reported that S. cerevisiae Gbp2p binds
specifically to single-stranded TG1-3 DNA in
vitro (12, 13). As shown in Fig. 1a,
cdc13-1 cells could not grow at 30 °C, whereas the same
cells expressing GBP2 grew to colonies. Expression of
GBP2 cannot complement the cdc13-1 phenotype at
37 °C and was not sufficient to complement the cdc13
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
and
subunits (8, 9), Cdc13p (10, 11), Gbp2p (12, 13), hnRNPs (14-16), and Pot1 (17), have been identified
in vitro. Among these proteins, Oxytricha
and
subunits have been well characterized because of the abundance of
telomeres in this organism. The
subunit binds to the
G4T4G4T4
single-stranded end of the telomere, and the
subunit is required
for making the terminus-specific binding (8, 18, 19).
Oxytricha
- and
-like-binding proteins represent a
novel type of DNA-binding protein because their binding is extremely
salt-resistant. The binding of other single-stranded telomeric binding
proteins to telomeric DNA is relatively salt-sensitive. A protein Pot1
that shares partial sequence homology with the Oxytricha
subunit has been identified from human and Schizosaccharomyces
pombe (17). Mutation in S. pombe POT1 causes loss of
telomeric DNA and circulation of chromosomes. In S. cerevisiae, Cdc13p binds specifically to single-stranded
TG1-3 DNA in vitro and affects telomere function in vivo (10, 11, 20, 21). Interestingly, even though Cdc13p does not share sequence similarity with the ciliate proteins or Pot1, the binding regions of Cdc13p and ciliate proteins that interact with single-stranded telomeric DNA are conserved (22,
23).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(MATa his3-11 trp1-1
leu2::pLexAop6-LEU2
ura3-1::URA3- pLexAop8-LacZ) was used in
the two-hybrid method (30).
-32P]ATP (3000 mCi/mM,
PerkinElmer Life Sciences) using T4 polynucleotide kinase (New
England Biolabs) and subsequently purified from a 10% sequencing gel
after electrophoresis. To perform the assays, Gbp2p, its mutants, or
E. coli extracts harboring Tcg1 in Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 1 µg of heat-denatured
poly(dI-dC)) was mixed with 5 nM of 32P-labeled
TG43 DNA in a total volume of 15 µl. The reaction mixture was
incubated at room temperature for 10 min and then loaded directly onto
an 8% non-denaturing polyacrylamide gel already pre-run at 125 V for
10 min. Electrophoresis was carried out in TBE (89 mM Tris
borate, 2 mM EDTA) at 125 V for 105 min. The gels were
dried and autoradiographed.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
mutation (data not shown). Another single-stranded TG1-3
DNA-binding protein, the product of S. pombe TCG1 (thirteen complementation gene,
GenBankTM accession number AY007252), also complements
cdc13-1. Similar to Gbp2p, TCG1 encodes a
protein with two RRM motifs that binds single-stranded
TG1-3 DNA (Fig. 1, b and c). Plasmid
harboring TCG1 rescued the growth defect of
cdc13-1 at 30 °C but could not complement
cdc13-1 at 33 °C (Fig. 1d). It should be
noted that the above complementation is specific to single-stranded
telomeric DNA-binding proteins because other RRM-containing proteins
that do not bind single-stranded telomeric DNA, including Ssb1p, Pub1p, or Hrb1p, did not complement the growth defect of cdc13-1
at 30 °C (Fig. 1a, and data not shown).
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Fig. 1.
Single-stranded telomeric binding proteins
rescue the growth defect of the cdc13-1 mutant.
a, yeast W13a (cdc13-1), carrying plasmids
pSH380, pGAL-GBP2, or pGAL-HRB1, was spotted in 10-fold serial
dilutions on YC-Leu plates with the addition of 3% galactose and grown
at 25 °C (left) or 30 °C (right) until
colonies formed. b, domain structure of Tcg1 protein.
TCG1 encodes a polypeptide of 349 amino acids. Two RRMs are
indicated. c, Tcg1 binds single-stranded TG1-3
DNA. Tcg1 was purified as a GST fusion protein in E. coli.
Purified GST (0.6 µg) and GST-Tcg1 (1 µg) were analyzed on a 10%
SDS-polyacrylamide gel, and the Coomassie Blue-stained gel is shown.
Arrows indicate the positions of purified proteins
(left). Purified GST or GST-Tcg1 (200 nM) was
mixed with 5 nM 32P-labeled TG43 in 15-µl
reaction mixtures. The reactions were incubated at room temperature for
10 min and then subjected to gel shift assay. Lane 1 has no
extracts. An autoradiogram is shown on the right.
Position of the protein-DNA complex is indicated by an
arrow. d, TCG1 complements
cdc13-1 mutation. Yeast 4053-5-2a (CDC13), W13a
(cdc13-1), or the W13a-carrying plasmid pTCG1or pDB20
(vector) was spotted in 10-fold serial dilutions on YC plates and grown
at 25 °C (left), 30 °C (middle), or
33 °C (right) until colonies formed.
Gbp2p contains two RRM motifs. The RRM motif, an
~90-amino acid module, is used for RNA binding in many RNA-binding
proteins (33). The signature of the RRM motif is the two consensus
sequences, RNP1 and RNP2, located about 30 amino acids apart in the RRM
domain (Fig. 2a). These two
conserved regions are involved in making contacts with RNA. In U1 small
nuclear RNP, the Arg residue in RNP1 forms a salt bridge with the
phosphate of U1 RNA, whereas residues in RNP2 form hydrogen bonds with
the RNA (34). We changed both Arg-161 and Arg-259, the conserved Arg
residues within the two RRM motifs of Gbp2p, to Ala (Fig.
2a), and tested the effect on the single-stranded
TG1-3 binding activity in a gel mobility shift assay using
purified mutant proteins (Fig. 2, b and c). The
results indicate that the R259A mutation greatly reduced the binding of
Gbp2p to single-stranded TG1-3 DNA, whereas the R161A
mutation only partially inactivated Gbp2p. Mutation on a non-conserved
residue, Arg-127, did not affect the binding activity (data not shown).
These results indicate that Arg-161 and Arg-259 are important for
binding to a single-stranded TG1-3 DNA.
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Each Gbp2p binding mutant was tested for efficiency in complementing
the growth defect of cdc13-1 cells. The experiments were conducted in the presence of glucose or galactose, because
GBP2 expression was driven by the GAL1 promoter.
As shown in Fig 3a, whereas
wild-type GBP2 complemented cdc13-1 even under
the non-inducing condition (Leu + Glc, 30 °C) and
gbp2R161A complemented only under the inducing
conduction (
Leu + Gal, 30 °C), neither
gbp2R259A nor gbp2R161A,
R259A was able to complement cdc13-1 at
30 °C. These results suggested that complementation depends on the
single-stranded telomeric DNA binding activity.
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We investigated the effect of mutant or wild-type Gbp2p on the
cdc13-1-associated telomere-lengthening phenotype (35). In S. cerevisiae, middle repetitive sequences known as Y'
elements are found in the subtelomeric regions of most chromosomes. As shown in Fig. 3b, in CDC13/pSH380 cells a
XhoI digest produces a ~1.3 kbp fragment from the ends of
Y'-bearing chromosomes that contains ~950 bp of Y' and the terminal
tract of ~350 bp of TG1-3/C1-3A DNA.
However, the length of Y'-bearing telomeres in cdc13-1
cells appeared to be longer and more heterogeneous. Expression of the wild-type GBP2 or gbp2R161A, but not
gbp2R259A or gbp2R161A,
R259A, suppressed telomere lengthening in
cdc13-1 cells. Expressing Gbp2p in wild-type cells or cells
with gbp2 or gbp2
cdc13-1 mutations did
not affect the telomere length (data not shown). The effect of
expressing Gbp2p on the accumulation of single-stranded telomeric DNA
in cdc13-1 cells at 30 °C was also evaluated. The cdc13-1 cells, but not the wild-type cells, accumulate
abnormally high levels of single-stranded telomeric DNA at 30 °C
(Fig. 3c). By taking the single-stranded TG1-3
DNA level in cdc13-1 cells at 30 °C as 100%, the
single-stranded TG1-3 DNA level in wild-type
CDC13 cells, cdc13-1 cells overexpressing
wild-type GBP2, R161A, R259A, or R161A/R259A mutants is 26%, 45%,
50%, 102%, and 91%, respectively. Thus, expressing the wild-type
Gbp2p or R161A mutant moderately decreased the levels of
single-stranded telomeric DNA, whereas R259A or R161A/R259A mutants of
Gbp2p did not have this effect. These results demonstrate that the
single-stranded TG1-3 DNA binding activity of Gbp2p is
required for suppressing the telomere lengthening of
cdc13-1 cells and affecting the accumulation of
single-stranded telomeric tails.
The presence of single-stranded DNA has been speculated as being the signal for the DNA-damage checkpoint (29) because it is present at the replication fork when replication is incomplete. Moreover, single-stranded DNA is a common intermediate in many repair pathways, including double-strand break repair, excision repair, and recombination repair (36-38). Here we have shown that expressing two single-stranded TG1-3 binding proteins, S. cerevisiae Gbp2p and S. pombe Tcg1, complemented the defects of cdc13-1 cells. In addition, the single-stranded TG1-3 DNA binding activity of Gbp2p is required for this complementation. Because the cell cycle defect in cdc13-1 is dependent on the DNA-damage checkpoint, our results strongly suggest that the presence of single-stranded TG1-3 DNA in cdc13-1 at a non-permissive temperature sends the signal for the DNA damage checkpoint. In wild-type cells, the binding of Cdc13p to the telomeric tail may convey a message to sensor proteins of the DNA-damage checkpoint, thus marking telomeres different from broken DNA ends. In cdc13-1 cells, masking the arrest signal with other single-stranded TG1-3 DNA binding activity is necessary at non-permissive temperatures to circumvent the chromosome-surveillance mechanism and prevent cell cycle arrest.
A group of RRM-containing proteins that bind to single-stranded
telomeric DNA has been identified in several organisms. For examples,
human hnRNP A1, A2/B1, D, and E proteins and mouse hnRNP A2/B1 bind
specifically to G-strand telomeric DNA (14-16). In S. cerevisiae, Gbp2p and Nsr1p each have two RRMs and bind
specifically to single-stranded TG1-3 DNA in
vitro (12, 13) These RRM-containing proteins were shown to affect
different telomere functions. For instance, mouse cells deficient in
hnRNP A1 have telomeres shorter than those of cells expressing a normal
level of hnRNP A1, suggesting a role for hnRNP A1 in telomere
maintenance (39). Therefore, these RRMs containing telomeric
DNA-binding proteins might function both in RNA metabolism and at
telomeres. However, how these proteins affect telomeres remains unclear
(16). It is conceivable that these proteins exert their influence
through interaction with telomere-associated proteins. For example, the association between hnRNP A1 or D proteins and the telomerase might
affect telomere maintenance (39, 40).
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ACKNOWLEDGEMENTS |
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We thank members of the Institute of Biopharmaceutical Sciences for the help and support. We also thank Dr. S. C. Teng for reading the manuscript.
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FOOTNOTES |
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* This research was supported by National Science Council Grants NSC 91-2312-B-010-008, NSC 91-2311-B-010-004, Program for Promoting Academic Excellence of Universities Grant 89-B-FA22-2-4 (to J.-J. L.), and National Health Research Institute (Taiwan) Grants NHRI-GT-EX89S916C and NHRI-EX90-8916SC (to M. Y. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work.
To whom correspondence may be addressed. Tel.: 02-2826-7258;
Fax: 02-2822-0084; E-mail: jjlin@ym.edu.tw or
meychen{at}ym.edu.tw.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M208347200
1 S. Hahn, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
GST, glutathione S-transferase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
DTT, dithiothreitol;
MBP, maltose-binding protein;
RNP, ribonucleoprotein;
RRM, RNA recognition motif.
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