* Institute of General Microbiology, University of Bern, CH-3012 Bern, Switzerland; Section of Human Biology and Genetics,
University of Kaiserslautern, D-67653 Kaiserslautern, Germany; § Basel Institute of Immunology, CH-4005 Basel, Switzerland;
and
Institute of Gene Biology, Moscow 117 334, Russia
Exoribonucleases are important enzymes for
the turnover of cellular RNA species. We have isolated
the first mammalian cDNA from mouse demonstrated
to encode a 5-3
exoribonuclease. The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in
cytoplasmic mRNA turnover and pre-rRNA processing
similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast. Therefore, a key component of
the mRNA decay system in S. cerevisiae has been conserved in evolution from yeasts to mammals. The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex-containing substrates demonstrated in binding and hydrolysis experiments. mXRN1p is the first RNA turnover function
that has been localized in the cytoplasm of mammalian
cells. mXRN1p was distributed in small granules and
was highly enriched in discrete, prominent foci. The
specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have
a role in RNA turnover.
Most cellular RNA species are synthesized as precursors, which are processed to yield mature
RNA molecules. Those regions of the pre-RNA
not found in the final molecule have to be degraded to
avoid accumulation of unwanted RNA species. The turnover of RNA species, in particular mRNA, is important in
determining the levels and regulation of gene expression
(for review see Ross 1995 Xrn1p (for review see Kearsey and Kipling, 1991
Xrn1p has been suggested to be the major 5 Here we report the first isolation of mammalian cDNAs
demonstrated to encode an exoribonuclease active in RNA
turnover. mXRN1p is the structural and functional mouse
homolog of the S. cerevisiae Xrn1p exoribonuclease. Therefore, it is likely to be involved in mRNA turnover and
rRNA processing in mouse cells. mXRN1p localizes to cytoplasmic granules and is enriched in prominent foci. The purified mouse protein exhibits 5 Media and Genetic Methods
The methods used for growing and constructing S. cerevisiae strains (Sherman et al., 1982 S. cerevisiae Strains
The S. cerevisiae strain WDHY131 (MATa ura3-52 trp1 leu2 Table I.
Complementation of Sporulation Defect in S. cerevisiae xrn1, 1996
; Caponigro and Parker,
1996
). Furthermore, the spatial distribution of certain proteins is achieved by localized control of mRNA stability
(St. Johnston, 1995). Whereas specific cis- and trans-acting
factors are required for the regulated decay of specific
mRNAs (Brawerman, 1993
), a more general pathway for the degradation of unwanted RNA is likely to exist into
which different RNA species are funneled. Seminal work
(for review see Beelman and Parker, 1995
; Caponigro and
Parker, 1996
) in the yeast Saccharomyces cerevisiae has
identified deadenylation-dependent and -independent decay of mRNA, which requires several enzymatic activities
including decapping, endoribonuclease, poly(A) nuclease, and 3
-5
and 5
-3
exoribonuclease.
;
Heyer, 1994
) and Rat1p (also known as Tap1p, Hke1p,
Exonuclease 2; for review see Stevens, 1993
) are 5
-3
exonucleases from S. cerevisiae. These two enzymes are the
only examples of purified 5
-3
exonucleases in RNA
turnover in pro- and eukaryotes (for review see Stevens, 1993
) and share substantial sequence homology (see Fig.
2), yet both enzymes have functionally diverged. A primarily nuclear role for Rat1p has been suggested (Amberg et
al., 1992
; Kenna et al., 1993
; Henry et al., 1994
), whereas
Xrn1p acts and is localized in the cytoplasm (Hsu and
Stevens, 1993
; Henry et al., 1994
; Muhlrad and Parker, 1994
;
Muhlrad et al., 1994
; Heyer et al., 1995
).
Fig. 2.
Structure and evolutionary conservation of mouse mXRN1p. (A). Structural relation
of mXRN1p to other yeast and mammalian proteins. The domains of strongest homology are indicated as black boxes (I-III). An additional domain (IV) with intermittent but highly conserved
sequence stretches between mXRN1p, Xrn1p,
and ExoIIp is hatched. Numbers above the boxes
represent the percentage of amino acid identity
to mXRN1p. References to the sequences are found in Materials and Methods. (B) Phylogenetic tree of mXRN1p and related proteins.
M.m., M. musculus; S.c., S. cerevisiae; S.p., S. pombe; aa, amino acids.
[View Larger Version of this Image (17K GIF file)]
-3
exoribonuclease in cytoplasmic mRNA turnover (Stevens, 1978
,
1980
) and is active in deadenylation-dependent and -independent pathways (Caponigro and Parker, 1996
). The
analysis of XRN1 mutants suggested a role in RNA turnover of pre-rRNA (Stevens et al., 1991
; Henry et al., 1994
)
and mRNA (for review see Beelman and Parker, 1995
;
Caponigro and Parker, 1996
; Jacobson and Peltz, 1996
). In
addition to molecular defects in RNA metabolism, the mutants exhibit pleiotropic phenotypes including slow
growth, meiotic arrest, and defects in microtubule-related processes (for review see Heyer, 1994
). Therefore, it is not
surprising that this gene has been isolated in several different screens. Xrn1p (Larimer and Stevens, 1990
) is also
known as Sep1p (Kolodner et al., 1987
; Tishkoff et al.,
1991
), Stp
p (Dykstra et al., 1990
, 1991), Kem1p (Kim et al.,
1990
), Rar5p (Kipling et al., 1991
), and Ski1p (for review see
Wickner, 1996
). It is unclear whether all mutant phenotypes
are the consequence of the RNA metabolism defects.
-3
exoribonuclease activity and a substrate preference for RNA G4 tetraplex-
containing substrates in binding and hydrolysis over a
monomeric RNA substrate of the same sequence. This
specificity was not previously identified for S. cerevisiae
Xrn1p. The mXRN1p exonuclease activity preferred RNA
substrates over DNA substrates, either G4 or monomeric.
This suggests that RNA G4 tetraplex structures may occur
in vivo, possibly with a role in RNA turnover.
Materials and Methods
) and media for S. cerevisiae (Sherman et al., 1982
; Bähler
et al., 1994
) have been described. To test sensitivity to benomyl, S. cerevisiae cultures were grown in SD-ura medium, and the titer was adjusted to
2 × 107 cells/ml. 3 µl of cells from serial 10-fold dilutions were spotted on
plates containing 0 or 15 µg/ml benomyl. Plates were incubated for 2 d at
30°C and photographed.
1 his3
200
pep4::HIS3 prb1-
1.6R can1R xrn1
::LEU2) has been described (Bashkirov et al., 1995
). For the meiosis experiment we used S. cerevisiae strains
that are isogenic derivatives of the wild-type isolate S. cerevisiae SK-1 (Kane and Roth, 1974
). The parent strain derives from a single spore, and
marker systems have been developed in N. Kleckner's laboratory (Harvard University, Cambridge, MA), who kindly supplied basic strains.
These strains show efficient and fast sporulation (see Table I). WDHY187
(MATa/MAT
ho::LYS2/ho::LYS2 lys2/lys2 ura3/ura3 leu2::hisG/leu2::
hisG his4B/his4X XRN1/xrn1
::URA3), WDHY213 (MATa/MAT
ho::
LYS2/ho::LYS2 lys2/lys2 ura3/ura3 leu2::hisG/leu2::hisG his4B/his4X
can1R/CAN1 xrn1
::URA3/xrn1
::LEU2), and WDHY344 (MATa/MAT
ho::LYS2/ho::LYS2 lys2/lys2 ura3/ura3 leu2::hisG/leu2::hisG his4B/his4X
can1R/CAN1 xrn1
::ura3/xrn1
::LEU2) were constructed for this study. A spontaneous mutation inactivating the URA3 gene disrupting XRN1 in
WDHY344 was isolated on medium containing 5-fluoro-orotic acid.
by mXrn1
Plasmids
The complementation plasmids are based on the S. cerevisiae CEN-ARS
plasmid YCp50 (see Bashkirov et al., 1995). pXRN1 (pWDH276) was derived from pJI82 (Kim et al., 1990
) containing a chromosomal fragment of
the XRN1 region by deletion of a vector SalI site and engineering a SalI site
just upstream from the ATG of the XRN1 open reading frame for efficient
further recloning. pmXrn1 (pWDH347) and pmXrn1
39 (pWDH348) were
derived from pXRN1 by substituting the XRN1 open reading frame with
the full-length mouse cDNAs using the promoter SalI site and the vector
HindIII site. pGALXRN1 is pRDK249 (Johnson and Kolodner, 1991
) in
which the XRN1 open reading frame is expressed under the control of
the GAL10 promoter. The mouse full-length cDNAs were cloned in
pRDK249, replacing the resident XRN1 open reading frame and resulting
in pGALmXrn1 (=pWDH349) and pGALmXrn1
39 (=pWDH350).
Vector control pGAL (=pWDH181) contains no open reading frame under the control of the GAL10 promoter of pRDK249.
Cloning of Mouse mXrn1
For PCR amplification, two oligonucleotides ending with EcoRI recognition sequences at their 5 ends (5
-GGAATTCCI(C/A)GIGCIAA(A/G) ATGAA(C/T)CA(A/G)CA-3
and 5
-GGAATTCATIAG(G/A)TCIGC(G/
A)T CIAGICC(G/A)TA-3
, where I refers to inosine) were synthesized.
These sequences correspond to highly conserved regions of S. cerevisiae
XRN1 (Kim et al., 1990
) and Schizosaccharomyces pombe Exo2 (Szankasi
and Smith, 1992
, 1996
), as well as S. cerevisiae RAT1 (Amberg et al., 1992
). The amino acid sequence encoded by the first oligonucleotide (PRAKMNQQ) corresponds to amino acid residues 90-97, and the sequence encoded by the second oligonucleotide (YGLDADLI) corresponds to residues 203-210 of the S. cerevisiae Xrn1 protein. PCR was performed with 5 ng of mouse testis cDNA as a template for 30 cycles of 5 s at 94°C, 15 s at
37°C, and 2 min at 72°C. The PCR products were analyzed on a 4% agarose gel (NuSieve®; FMC BioProducts, Rockland, ME), eluted from the
gel, and subcloned in M13mp19 for sequence analysis. A 363-bp PCR
product was used as a probe for the first round screening of a mouse testis
cDNA bank. Recombinant phages
511,
14, and
1620 were isolated
from a mouse testis cDNA library in
gt10 (BALB/c; Clontech, Palo Alto, CA),
2100 from a mouse testis cDNA library in
gt11 (BALB/C; Clontech), and
10,
1,
6,
4,
9, and
3 from a mouse thymus cDNA library
in
ZAP (B6/CBAF1J; Stratagene Inc., La Jolla, CA) using the probes indicated in Fig. 1 A. All cDNA clones displayed in Fig. 1 were sequenced
on both strands. Full-length cDNA constructs were reconstructed by joining inserts from
511 and
10 at their unique HpaI sites for pmXRN1 and
joining inserts from
511 and
1 for pmXrn1
39. Standard procedures
were used for DNA library screening, DNA hybridization, and sequencing. These sequence data are available from EMBL/GenBank/DDBJ under accession number X91617.
Purification of Mouse mXRN1p
S. cerevisiae strain WDHY131 (xrn1) bearing plasmid pGALmXrn1 was
induced by galactose for overproduction of mouse mXRN1p as described
(Johnson and Kolodner, 1991
). After harvesting and washing in 20 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, and 1 mM PMSF, the cells
were frozen in liquid nitrogen and stored at
70°C. 24 g of cells was
thawed on ice. All the steps were performed at 4°C. mXRN1p purification
was monitored by Western blot with a rabbit polyclonal anti-mXRN1p antibody. The cell slurry was diluted to 1.25 ml of buffer A containing 300 mM
NaCl per gram of cells. Buffer A is 20 mM Tris-HCl, pH 7.5, 1 mM
EDTA, 10% (wt/vol) glycerol, 10 mM
-mercaptoethanol, 0.2 mM PMSF,
2 mM benzamidine, 2 µM pepstatin A, and 2 µM leupeptin. Cells were disrupted in a bead beater (Biospec Products, Bartlesville, OK) with six
30-s pulses with 2 min of cooling between pulses. The glass beads were
washed twice with 10 ml buffer A/300 mM NaCl. Cell lysate and wash
were combined and centrifuged in a Ti45 rotor (45 min, 40 krpm at 4°C)
resulting in fraction I (15.7 mg/ml, 75 ml). Fraction I was loaded on a phosphocellulose column (P11; Whatman Inc., Clifton, NJ) equilibrated with
buffer A/300 mM NaCl at a flow rate of 70 ml/h. The flowthrough was collected as fraction II (100 ml, 10 mg/ml) and centrifuged at 8 krpm for 20 min to collect the precipitate. The supernatant (fraction III, 4 mg/ml) was
reapplied on a new P11 column preequilibrated in buffer A/300 mM NaCl.
After washing with buffer A/300 mM NaCl, proteins were eluted with 150 ml of buffer A/1 M NaCl. The eluting fractions containing mXRN1p were
pooled (35 ml) and concentrated on an ultra filtration unit (Centriprep100; Amicon, Beverly, MA) to result in fraction IV (0.9 mg/ml, 3.5 ml).
The conductivity of fraction IV was adjusted to 1 M NaCl and loaded on a
gel filtration column (Sephacryl S-200; Pharmacia, Piscataway, NJ; 2 cm2 × 70 cm) at a flow rate of 19 ml/h. mXRN1p-containing fractions were dialyzed against buffer B containing 20 mM Tris-HCl, pH 7.5, 0.1 mM
EDTA, 1.0 mM DTT, 500 mM NaCl, and 60% (wt/vol) glycerol and
stored at
20°C (fraction V; 0.02 mg/ml, 0.32 ml). All experiments were
performed using fraction V.
Protein Methods and Antibodies
Glass bead extracts of total S. cerevisiae protein were done as described
(Johnson and Kolodner, 1991). Protein extracts and fractionation from
adult mouse testis cells was performed as described (Dignam, 1990
). Proteins were separated on 8% SDS-PAGE and either stained with Coomassie brilliant blue or analyzed by Western blotting. As first antibodies,
we used affinity-purified rabbit anti-mXRN1p antibodies (0.2 µg/ml) or
the Ig fraction of rat anti-mXRN1p antibodies (1:5,000 dilution), which
recognize mouse mXRN1p but not S. cerevisiae Xrn1p. Signals were detected using HRP-conjugated anti-rabbit or anti-rat IgG antibodies (1:
4,000 or 1:7,500 dilution, respectively) as secondary antibodies in the ECL
system (Amersham Corp., Arlington Heights, IL) according to manufacturer's instructions. To visualize the S. cerevisiae Xrn1p on the same blot,
the anti-S. cerevisiae Xrn1p mouse mAb H8 (Heyer et al., 1995
) (1 µg/ml)
was used after detecting mouse mXRN1p. Xrn1p signals were generated
using HRP-conjugated rabbit anti-mouse Ig antibodies (1:4,000 dilution;
ECL system; Amersham Corp.).
The anti-mouse mXRN1p antibodies were generated against a His(6)
fusion to amino acids 1,132-1,719 of mXRN1p, which was overexpressed in Escherichia coli using the pT7-7 system (Tabor and Richardson, 1985).
The protein was purified by Ni++ chelate affinity chromatography
(Qiagen, Inc., Chatsworth, CA) according to the manufacturer's instructions followed by preparative SDS-PAGE and electroelution of the protein band from the gel slice. 400 and 100 µg of purified antigen were injected into rabbits or rats, respectively, and the immune response was
boosted three times. Antibodies were affinity purified from serum on
His(6)-mXRN1p(aa 1,132-1,719) containing nitrocellulose strips as described (Pringle et al., 1991
).
Biochemical Methods
Exoribonuclease activity was assayed essentially as described (Stevens,
1978, 1980
; Käslin and Heyer, 1994
). 3.72 pmol (2.6 × 104 cpm/pmol) of
[32P]GTP-labeled T7 in vitro transcript of 367 nt mouse
-actin was used
as a substrate in reactions containing 100 fmol of mXRN1p.
G4 tetraplex DNA or RNA was prepared from deoxy-oligonucleotide
(5-TATGGGGGAGCTGGGGAAGGTGGGATTT-3
; called GL) or
the same sequence of ribo-oligonucleotide (rGL) and 5
end-labeled with T4 polynucleotide kinase using [
-32P]ATP essentially as described (Frantz
and Gilbert, 1995
). For binding assays (20 µl), 0.3 fmol of 5
32P-labeled
tetraplex RNA (rGL[G4]), tetraplex DNA (GL[G4]), or single-stranded
oligo (rGL[SS]) were incubated with 0.05-200 fmol protein at 4°C for 20 min in binding buffer (20 mM Hepes, pH 7.5, 100 mM KCl, 10% glycerol,
1 pmol unlabeled oligonucleotide TP-S 5
-TGGACCAGACCTAGCA3
, and 1 µg poly [dI-dC]: poly[dI-dC]) and run on 6% polyacrylamide gel
(Sen and Gilbert, 1988
; Liu and Gilbert, 1994
). The dried gels were quantified using a PhosphorImager. For nuclease assays, 20 fmol of the
GL(G4), rGL(G4), GL(SS), and rGL(SS) substrates and 2.5 fmol of
mXRN1p unless otherwise specified were used in binding buffer supplemented with 3 mM MgCl2 without the unlabeled TP-S oligonucleotide.
The reaction was carried out at 37°C. The extent of hydrolysis was determined by quantification of the dinucleotide first cleavage product on a
PhosphorImager expressed as fmol of cleaved substrate.
mXRN1p was immunoprecipitated from fraction V using magnetic beads coated with sheep anti-mouse IgG1 (Fc) (30 mg/ml; Dynabeads M-450; Dynal Inc., Great Neck, NY) coupled to 20 µg of the anti-Xrn1p mAbs H8 or B4 (Holler et al., 1994) by overnight rocking at 4°C in 500 µl PBS (0.15 M NaCl and 0.01 Na-phosphate, pH 7.4) containing 0.1% BSA. Both anti-Xrn1p mAbs are of the IgG1 subtype; mAb-H8 recognizes both S. cerevisiae Xrn1p and mouse mXRN1p, whereas mAb-B4 is specific for the S. cerevisiae Xrn1 protein. After coupling, the beads were washed three times in PBS containing BSA and once in PBS containing BSA, 0.5 M NaCl, and 5% (wt/vol) glycerol. Each sample of Dynabeads loaded with primary and secondary antibodies, or only with primary antibody (control), was divided on two parts and separately incubated with 40 ng of mXRN1p (fraction V) in 500 µl of PBS containing BSA, 0.5 M NaCl, and 5% (wt/vol) glycerol for 12 h at 4°C. After washing the Dynabeads four times in the same buffer and once in 10 mM Tris-HCl, pH 8.8, the binding between primary and secondary antibodies was broken by treatment with 25 µl of freshly prepared 100 mM triethylamine, pH 11.5, for 10 min at 4°C. The immunoprecipitate was neutralized with 2 µl of 1 M Tris-HCl, pH 7.5. 5 µl of the immunoprecipitate was used for the binding reaction with 32P-labeled G4 DNA as substrate, and another 5 µl was used for immunoblotting for detection of mXrn1p. Under these conditions the exonuclease function of mXRN1p lost its activity (data not shown).
Immunofluorescence Methods
Cells were grown in DME (Life Technologies, Gaithersburg, MD) and
15% FCS on clean glass slides to 80% confluence. Benomyl was added to
the culture medium at 40 µg/ml for 5 h, while cold-treated cells were kept
at 4°C for 10 h. The efficacy of the drug treatment or the cold treatment
was demonstrated by the reduced immunofluorescence using anti-tubulin
antibodies (see Fig. 9). Cells were washed in PBS and fixed for 10 min in
4% formaldehyde, 0.02% glutaraldehyde, and 0.2% Triton X-100/PBS.
After subsequent washing, cells were simultaneously immunostained (Giese
et al., 1995) with rat anti-tubulin antibodies (Serotec Ltd., Oxford, UK;
second antibody was an FITC conjugate, Sigma Chemical Co., St. Louis,
MO) and rabbit anti-mouse mXRN1p antibodies (second antibody was a
Cy3 conjugate, Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA). Immunostaining of the actin cytoskeleton was performed with coumarin-phaloidin (Sigma Chemical Co.). Antibodies against vimentin were
kindly provided by G. Giese (MPI Cell Biology, Ladenburg, Germany).
The anti-mXRN1p antibodies used for immunofluorescence were the
same affinity-purified antibodies used in Western blot analysis (see Fig.
6). Fig. 6 demonstrates their specificity for mXRN1p. Finally, preparations were embedded in antifade solution (Vector Laboratories, Inc., Burlingame, CA) and analyzed using a fluorescence microscope (Axioskop;
Carl Zeiss, Inc., Thornwood, NY) equipped with single-band pass filters
for excitation of red, green, and blue fluorescence (Chroma Technologies
Corp., Brattleboro, VT). Images of high magnification and resolution
were obtained using a black and white CCD camera controlled by ISIS
fluorescence image analysis software (METASystems, Altlussheim, Germany). The number of foci was counted in 50 cells using a size cutoff of
300 nm; given are mean numbers ± 1 SD. The diameter of foci was determined in at least 27 foci from two independent experiments; given is the
mean diameter ± 1 SD. Measurements were performed on digitized and
enhanced images using the measurement option of the ISIS fluorescence
image analysis software package.
Computer Analysis
Homologies of mXRN1p to other proteins were detected in a FASTA
search. The sequences were aligned, and the phylogenetic tree was generated with the Growtree program using the Kimura protein distance algorithm. The identity between individual homology boxes was scored in
GAP alignments (gap weight = 3, length weight = 0.1). The sequences
were S. cerevisiae Xrn1p (Kim et al., 1990) and Rat1p (Amberg et al.,
1992
), S. pombe Dhp1p (Sugano et al., 1994
), ExoIIp (Szankasi and Smith,
1996
), and mouse Dhm1p (Shobuike et al., 1995
). All programs were implemented in the GCG software package (version 8.0; Genetics Computer
Group, Inc., Madison, WI) (Devereux et al., 1984
).
Mouse cDNAs Encoding an Exoribonuclease
Mouse cDNAs deriving from the mammalian homolog of
S. cerevisiae XRN1 (Fig. 1) have been identified by a PCRbased approach. A full-length cDNA of 5,497 nt was assembled, and an RNA consistent with this predicted size
was detected in Northern blots of RNA isolated from
mouse tissues (data not shown). Two cDNA variants were
recovered, mXrn1 and mXrn139. mXrn1
39 carried an
in-frame 39-bp deletion leading to a predicted protein
product smaller by 13 amino acids (Fig. 1). PCR analysis
of genomic DNA and cDNA derived from testis suggests
that both cDNAs represent splice variants of a single-copy
gene and that the deleted sequence in mXrn1
39 corresponds to an optional exon (data not shown). The DNA
sequence (accession number X91617, not shown) predicts
a 5,157-bp open reading frame for mXrn1 with a coding
potential for a 194.2-kD protein composed of 1,719 amino
acid residues and, for mXrn1
39, a 5,118-bp open reading
frame potentially encoding a 192.9-kD protein. The putative initiation codon for both cDNAs is located in a region
with highly significant homology to the NH2-terminal region of the homologous genes (Kim et al., 1990
; Amberg et
al., 1992
) (see Fig. 2 A). In addition, the sequence around
this ATG initiation codon (AAAATGGGA) fits well to
the consensus proposed by Kozak (1991)
with a purine
and a guanosine at positions
3 and +4, respectively.
Given the additional fact that the cDNAs exhibit biological activity (Figs. 3-5 and below), we conclude that the indicated ATG is highly likely to be the start codon of the mXrn1 gene. The 3
end of both cDNAs is characterized
by a relatively short oligo(A) tail of between 8 and 22 residues in six different clones (Fig. 1). Short poly(A) tails
have been suggested to be of importance for the regulation
of expression of several eukaryotic genes (Baker, 1993
).
Database searches revealed homologies to the protein sequences of the genes used for the PCR cloning strategy (see Materials and Methods), S. cerevisiae XRN1 and S. pombe exo2, and to a group of related proteins and predicted proteins from S. cerevisiae, S. pombe, and mouse (Fig. 2). No other significant homologies were identified. The phylogenetic relationship between the individual sequences (Fig. 2 B) suggests the existence of two subfamilies of related proteins with demonstrated or suspected exonuclease activity. In both subfamilies, the two yeast sequences show a slightly closer relationship to each other than to the mouse sequence.
Mouse mXRN1p Is Functional in S. cerevisiae
The sequence data suggested that the mouse cDNAs derive from a homolog of the yeast genes, and we sought
functional evidence to support this notion. To this end, we
constructed plasmids placing the two mouse cDNA variants under the control of the cognate S. cerevisiae XRN1
promoter to yield pmXrn1 and pmXrn139 for the long
and short variants, respectively. These plasmids together with the equivalent S. cerevisiae construct (pXRN1) were
used to assay for complementation of defects caused by a
deletion of the XRN1 gene in S. cerevisiae (see Figs. 3-5).
xrn1 cells exhibit pleiotropic phenotypes caused by defects in RNA turnover (for review see Stevens, 1993
; Caponigro and Parker, 1996
) and in the cytoskeleton (Kim
et al., 1990
; Interthal et al., 1995
). Both mouse cDNAs
complemented the slow-growth phenotype almost as efficiently as the cognate plasmid-borne gene (doubling times:
vector control, 209 min; pXRN1, 148 min; pmXrn1, 166 min; pmXrn1
39, 170 min; see also Fig. 3 for a semiquantitative test). Moreover, the benomyl hypersensitivity of the
S. cerevisiae mutant was also complemented to a large extent, but not as efficiently as by the S. cerevisiae XRN1
gene (Fig. 3).
A striking phenotype of xrn1 cells is a quantitative
meiotic prophase arrest in pachytene leading to highly reduced sporulation (Table I) (Bähler et al., 1994
; Tishkoff
et al., 1995
). This phenotype is complemented by both
mouse cDNAs essentially to the same extent as by the S. cerevisiae gene (Table I).
One RNA turnover defect in xrn1 cells is the accumulation of a fragment of the internal transcribed spacer of
pre-rRNA, which is usually degraded during cytoplasmic
processing of the 20S precursor to the 18S RNA (Stevens
et al., 1991
) (Fig. 4 A). The accumulation of this fragment
can be visualized by Northern blot analysis in total RNA
of xrn1
cells (Fig. 4 B, lane 1) but not in xrn1
cells containing the wild-type gene on a plasmid (lane 2). Both
mouse cDNAs complemented this molecular defect of the
S. cerevisiae mutant to the same extent as the cognate gene
(lanes 3 and 4).
mRNA turnover defects in xrn1 cells are signaled,
for example, by the accumulation of certain mRNAs as
poly(A)
species (Hsu and Stevens, 1993
). A large XRN1dependent effect can be observed for the RP51A mRNA,
whereas only a small effect can be documented for ACT1
mRNA (Fig. 5), consistent with previous observations (Hsu
and Stevens, 1993
). Both defects can be complemented by
the cognate wild-type gene or by both mouse cDNAs, essentially to the same extent (Fig. 5).
Purification and Characterization of mXrn1p
The open reading frame discovered in the mouse cDNA
predicted a protein of 194 kD, and a polypeptide of this
size can be visualized by immunoblot analysis in S. cerevisiae cells overexpressing the cDNAs from the regulated
GAL10 promoter (Fig. 6 A, lanes 3 and 4). Moreover, a
polypeptide of identical size can be detected in cytoplasmic protein extracts from mouse testis (lane 5). This demonstrates that the mouse gene from which we derived the
cDNAs encodes the predicted protein in vivo. No immunoreactive bands of this size were detected in high salt extracts of nuclei (data not shown), consistent with the localization of the protein in the cytoplasm (see below). In
addition, this analysis demonstrated the specificity of the
anti-mXRN1p antibodies because they recognize only the
mXRN1p band in protein extracts from mouse testis (Fig. 6 A) and other tissues as well as from mouse E10 cells
(data not shown). To obtain direct biochemical evidence
that mouse mXRN1p exhibits exoribonuclease activity, we
purified the protein (Fig. 6 B) and demonstrated in vitro
exoribonuclease activity (Fig. 7 A). Because both mouse
cDNA variants (mXrn1 and mXrn139) were found to
have identical biological activity in all interspecies complementation assays (Figs. 3-5), we decided to purify and
characterize only the longer version of mouse mXRN1p.
A nuclear role for Xrn1p has been proposed based on
the specificity of the enzyme for G4 tetraplex-containing
DNA substrates (Liu and Gilbert, 1994; Liu et al., 1995
).
mXRN1p exhibited a substrate binding specificity for G4
tetraplex-containing DNA and RNA substrates (Fig. 7 B).
In these experiments (Fig. 7, B-D) we used the same sequence oligonucleotides either as RNA (rGL) or as DNA
(GL) in their monomeric form (rGL[ss], GL[ss]) or in their G4 tetraplex form (rGL[G4], GL[G4]) after forming
the tetraplex in vitro and purifying the tetraplex substrate
as described in Materials and Methods. In binding experiments, the equilibrium constants derived from Scatchard
analysis of the data shown in Fig. 7 B showed a higher affinity of mXRN1p for the G4 RNA substrate (Keq = 2.95 ± 0.32 × 1010 M
1) than for the G4 DNA substrate (Keq = 1.26 ± 0.24 × 1010 M
1) or the monomeric RNA oligonucleotide of the same sequence (Keq = 2.57 ± 0.34 × 109
M
1). These differences are small but significant, as indicated by the nonoverlapping standard deviations. Kinetic
analysis of the exonuclease activity using G4 tetraplex and
monomeric RNA as well as DNA substrates revealed
more striking differences (Fig. 7 C). mXRN1p hydrolyzed
the first cleavage in the RNA substrates (G4 tetraplex and
monomeric) with a biphasic kinetics, exhibiting a fast early
and a slower late component. Both DNA substrates were
hydrolyzed with a more uniform and significantly slower
kinetics than the RNA substrates. In particular, a large difference was apparent between the RNA and DNA G4 tetraplex substrates. During the first reaction phase, hydrolysis of the RNA G4 substrate was at least 14 times faster
than that of G4 DNA (Fig. 7 C). The kinetics data of the
hydrolysis also demonstrate a significant preference of
mXRN1p for the G4 tetraplex versus monomeric RNA
substrate (Fig. 7 C) consistent with the quantitative binding studies (Fig. 7 B).
A protein titration of mXRN1p using G4 tetraplex DNA
and RNA substrates is shown in Fig. 7 D. These data visualize again the preference of mXRN1p for RNA substrates.
In addition, the gel shows the position of the first cleavage
product (CP in Fig. 7 D), a radiolabeled two-nucleotide
product. This was also shown by further high resolution
gel electrophoresis using appropriate standards (data not
shown). Because the substrate has been labeled at its 5
end, this analysis also demonstrates that mXRN1p is a 5
-
3
exonuclease.
Several lines of evidence suggest that the exonuclease
activity and the substrate specificity for G4 tetraplex substrates is intrinsic to mXRN1p and not due to a minor contaminant. First, the 5-3
exonuclease activity of mXRN1p
and its enzymatic characteristics is consistent with the biochemical analysis of the homologous enzymes from S. cerevisiae (Stevens, 1980
; Johnson and Kolodner, 1991
, 1994
)
and S. pombe (Szankasi and Smith, 1992
; Käslin and Heyer, 1994
), which also possess 5
-3
exonuclease activity. Second, the mouse enzyme has been purified from a
S. cerevisiae strain deleted for the endogenous XRN1
gene, which encodes Xrn1p, the major 5
-3
exonuclease
activity in S. cerevisiae cells. Third, the amount of protein
added in the nuclease experiments (Fig. 7, A and C; see
Materials and Methods) was small, working at excess of
substrate. Any contaminant would have to be an unusually
active nuclease, presently unknown in S. cerevisiae. In
side-by-side experiments using mXRN1p and S. cerevisiae
Xrn1p, the mouse enzyme exhibited higher specific activity than the yeast enzyme (Bashkirov, V.I., and W.-D.
Heyer, unpublished data). Fourth, the amount of protein
added in the binding experiments (Fig. 7 B) was low,
showing 50% binding of 0.3 fmol of substrate at only 2.3fold excess of mXRN1p (0.68 fmol as calculated from Fig.
7 B). This quantitatively excludes a minor contaminant being responsible for the G4 tetraplex binding activity. Fifth,
an immunoprecipitation experiment (Fig. 8) demonstrated
that the G4 tetraplex specificity was intrinsic to mXRN1p
by an independent method. Using two anti-Xrn1p mAbs, one cross-reacting with mXRN1p (H8) and the other (B4)
not cross-reacting with the mouse protein, we demonstrated that only H8, the antibody recognizing the mouse
protein, could immunoprecipitate mXRN1p (Fig. 8 A) and
that the immunoprecipitate formed a specific complex with G4 tetraplex DNA (Fig. 8 B). Sixth, the observed
coupling of the binding and the cleavage reaction on several G4-containing substrates (Fig. 7, B and C, and data not
shown) argues that both activities were mediated by one
protein. Finally, the temperature optimum of the mXRN1p
nuclease activity was determined to be 37°C, typical for a
mammalian but atypical for a yeast enzyme. The S. cerevisiae Xrn1p nuclease activity showed an optimum at 30°C.
mXRN1p Has Two Types of Localization in Mammalian Cytoplasm
mXRN1p immunostaining revealed a general granular signal and an enrichment in a number of discrete, prominent foci (>300 nm) in the cytoplasm of mouse E10 cells (Fig. 9) and skin fibroblast cell lines as well as in rat RBL-1 and human HeLa cells (data not shown). We focus here on mouse E10 cells because their large and well-defined cytoplasm allowed more precise definition of the sublocalization. However, essentially the same conclusions are reached from the analysis of the other cell types. The cytoplasmic foci and the general granular staining did not appear with preimmune control antibodies (Fig. 9) or with other antibodies against cytoskeletal components including tubulin (Fig. 9), actin, and vimentin (data not shown). Double and triple immunofluorescence experiments using antibodies against mXRN1p and against cytoskeletal components concomitantly demonstrated at the light microscopic level the general absence of tubulin, actin, or vimentin from the mXRN1p containing foci (data not shown). This is consistent with the immunofluorescence data shown in Fig. 9. In mouse E10 cells, an average of 10.5 ± 4.1 mXRN1p-containing foci were detected per cell, with an average diameter of 570 ± 113 nm. Similar numbers of foci were observed in mouse skin fibroblasts (11.3 ± 8.5) and in HeLa cells (11.1 ± 4.3).
The general granular staining (Fig. 9) consists of smaller foci (<250 nm) resolved at high contrast and resolution only by digital image analysis (data not shown). Addition of benomyl, a drug that inhibits microtubule polymerization and effectively destroys microtubular structures, essentially abolished the cytoplasmic microtubuli network as expected and also the general cytoplasmic localization of mXRN1p but not the localization in foci (Fig. 9 d). Similarly, cold treatment (Fig. 9 c), which is known to destroy microtubular structures, reduced the cytoplasmic microtubular immunofluorescence as well as the general granular cytoplasmic but not the localization in foci of mXRN1p. The similarity between the cytological appearance of mXRN1p staining in Fig. 9, c and d, suggests that the benomyl effect is not a drug-related artifact.
The cytoplasmic localization of mXRN1p in mammalian
cells is consistent with the localization of Xrn1p in S. cerevisiae (Heyer et al., 1995), which could not provide the
structural details seen here for the small size of the yeast
cells. There is no evidence by immunofluorescence or cell
fraction studies that Xrn1p in S. cerevisiae occurs in the
nucleus (Heyer et al., 1995
). Equally, mXRN1p in mouse
has only been identified in the cytoplasm of tissue culture
cells or in testis tissue preparations by use of immunofluorescence and cell fraction techniques (Fig. 9; Scherthan,
H., V.I. Bashkirov, and W.-D. Heyer, unpublished data).
Although the data presented here show no positive evidence for nuclear localization of mXRN1p, it will be very
difficult to totally exclude that small amounts of mXRN1p
are present in the nucleus. A detailed study using confocal laser scanning microscopy will help resolve this issue.
mXRN1p Is the Mouse Homolog of the Major S. cerevisiae Cytoplasmic Exoribonuclease Xrn1p
S. cerevisiae Xrn1p is likely to be the major cytoplasmic exoribonuclease for RNA turnover of mRNA, the internal
transcribed spacer of pre-rRNA, and possibly other RNA
species (for review see Stevens, 1993; Caponigro and Parker,
1996
; Jacobson and Peltz, 1996
). The conservation of the
molecular structure (Fig. 2) and the cellular functions (Figs.
3-5) gives compelling evidence that mXRN1p of mouse is
the Xrn1p homolog of higher eukaryotes. This strongly
suggests a similar function for mXRN1p in mouse cells. This is consistent with the evolutionary conservation of
the general mRNA structure (cap, poly(A) tail) and of the
pre-rRNA structure and processing (Eichler and Craig,
1994
; Venema and Tollervey, 1995
).
To date, no difference in biological activity of the two
mXRN1p variants in mouse (mXrn1 and mXrn139) has
been detected, and the biological significance of the two
forms remains unclear. They may have variable distribution and relative abundance in different tissues.
It is presently unclear whether mXRN1p is related to a
5 exoribonuclease partially purified from cytoplasmic
mouse sarcoma cell extracts (Coutts and Brawerman,
1993
). However, the hydrolysis products of both activities
are somewhat different. mXRN1p and its S. cerevisiae homolog, Xrn1p (Liu and Gilbert, 1994
), make a dinucleotide as a first cleavage product and mononucleotides thereafter, whereas the mouse sarcoma activity produces
mono-, di-, and trinucleotides (Coutts and Brawerman,
1993
). mXRN1p is obviously unrelated to the ~37-kD 3
-5
exoribonuclease purified from human cells (Caruccio and
Ross, 1994
).
Cellular Roles of Mouse mXRN1p and S. cerevisiae Xrn1p: Nuclear versus Cytoplasmic
Much attention has been given to possible biological functions of G4 tetraplex structures potentially occurring at telomeric DNA (Sen and Gilbert, 1988; for review see Williamson, 1994
). In particular, it was suggested that Xrn1p of
S. cerevisiae plays a role in nuclear DNA metabolism as an
endonuclease acting on G4 tetraplex substrates (Liu and
Gilbert, 1994
). However, neither Xrn1p nor mouse mXRN1p
are endonucleases, but rather are 5
-3
exonucleases producing generally mononucleotide products with a first
cleavage product of two nucleotides (Fig. 7 d; Stevens, 1980
;
Liu and Gilbert, 1994
; Bashkirov, V.I., and W.-D. Heyer, unpublished observation).
The mutant phenotypes in S. cerevisiae (see Introduction
for references) and the in situ localization in yeast (Heyer
et al., 1995) and mouse (Fig. 9) suggested a cytoplasmic
rather than a nuclear role for the enzymes; therefore, we
tested the G4 tetraplex specificity of mouse mXRN1p on
RNA and DNA substrates of the same sequence using the
corresponding monomeric oligonucleotides as further controls. Previous work on S. cerevisiae Xrn1p (Liu and Gilbert, 1994
) was not quantitative and examined G4 DNA
substrates but did not analyze RNA substrates. The results
of this analysis (Fig. 7, B-D) clearly showed a high specificity of mXRN1p for RNA over DNA substrates and a
strong preference for the G4 RNA substrate versus monomeric substrate. Similar substrate preferences were found
with the S. cerevisiae Xrn1 protein (Bashkirov, V.I., and
W.-D. Heyer, unpublished results).
The pachytene arrest of cells lacking this protein (Bähler et al., 1994; Tishkoff et al., 1995
) was interpreted as a
result of a role of this protein in nuclear DNA metabolism
(Liu and Gilbert, 1994
). Other molecular defects manifest
in xrn1 cells (see Introduction) may also indirectly lead to
this meiotic arrest phenotype. Moreover, it is unclear
whether G4 tetraplex DNA structures form at all during
meiotic prophase. In conclusion, all available evidence regarding in situ localization, substrate specificity, and mutant phenotype in S. cerevisiae suggests that these proteins
are cytoplasmic in both organisms, consistent with a role in
cytoplasmic RNA turnover.
Implications of the Mouse mXRN1p Specificity for G4 RNA Tetraplex Substrates
G4 tetraplex structures were first noted in vitro by use of
RNA substrates (Zimmermann et al., 1975) requiring only
as few as four contiguous G residues (Cheong and Moore,
1992
). They are no less likely to occur in vivo than G4 tetraplex DNA structures (Kim et al., 1991
). The occurrence
of cellular enzymes such as mouse mXRN1p (Fig. 7) and
Xrn1p (Bashkirov, V.I., and W.-D. Heyer, unpublished
observation) with high specificity for G4 tetraplex RNA
substrates suggests that these structures may actually occur in vivo. Biochemical evidence suggested G4 tetraplex
RNA formation as a mechanism for the dimerization of
the HIV-1 genomic RNA (Sundquist and Heaphy, 1993
).
A G-rich region has been implicated in the endonucleolytic cleavage of the human insulin-like growth factor II
mRNA (Scheper et al., 1995
). However, the importance of
forming a stem-loop structure with a C-rich strand (Scheper et al., 1995
) makes involvement of a G4 tetraplex
structure less likely.
If G4 tetraplex RNA occurs in vivo, what could be the
functional significance? Given the role of Xrn1p and, by
implication, of mXRN1p in RNA turnover, one might
speculate on a functional role of this RNA structure in
RNA metabolism, specifically RNA turnover. From the
enzymological properties of mXRN1p, it is evident that it
will bind to G4 tetraplex-containing RNAs with preference because the Keq shows at least a 10-fold difference,
and that it will hydrolyze G4 RNA with an initial rate 15 times faster than monomeric RNA. However, the progression of the exonuclease activity is clearly slowed by the G4
tetraplex structure as shown by using 3-end labeled substrates (Bashkirov, V.I., and W.-D. Heyer, unpublished
observation). In vivo, G-rich sequences have been found
to stabilize RNA sequences 3
but not 5
of the G stretch (Vreken and Raué, 1992
; Decker and Parker, 1993
; Muhlrad et al., 1995
), consistent with Xrn1p being the relevant
5
-3
exoribonuclease in RNA turnover. However, G4 tetraplex formation might not be required for this effect because the Xrn1p already pauses at G-rich sites on monomeric (i.e., non-G4) substrates (Johnson and Kolodner, 1994
). Addition of a G stretch upstream of the AUG initiation codon greatly destabilized the PGK mRNA, reducing the half-life from 35 to 7 min (Muhlrad et al., 1995
).
This effect has not been found for the MFA2 mRNA or with
an insertion of a G-rich sequence downstream of the stop
codon in the PGK mRNA (Decker and Parker, 1993
;
Muhlrad et al., 1994
, 1995
). This differential effect could be explained by the enzymatic properties of Xrn1p/
mXRN1p by suggesting that, in the former case (general
destabilization by G stretch insertion), G tetraplex formation occurs. This would attract the nuclease activity to hydrolyze the sequence 5
to the G4 tetraplex. In the latter
case (no general destabilization), no G4 tetraplex formation occurs, leaving the overall half-life of the full-length mRNA unchanged but stabilizing, as in the former case,
the sequence 3
of the G insertion.
It has been proposed that decapping of mRNA is the
major control point for mRNA decay (Caponigro and
Parker, 1996). Apparently the only gene encoding such an
activity has been identified in S. cerevisiae, and strains lacking this activity exhibit a growth impairment (Beelman et
al., 1996
), as do S. cerevisiae xrn1
cells. Besides a control
point by a decapping enzyme, a second control point exerted by the S. cerevisiae Xrn1p or correspondingly by the
mouse mXRN1p exoribonuclease is suggested here. Although Xrn1p has been shown to be more active on decapped RNA, the residual activity on capped RNAs (Stevens, 1978
) paired with the G4 specificity provides substance
for such a control point. RNA G4 tetraplex formation is a
slow process compared with most other nucleic acid annealing processes (Sundquist and Heaphy, 1993
). Therefore, occurrence of G4 tetraplex structures is correlated to
the lifetime of an RNA. A possible model for the role of
G4 tetraplex RNA structures is that they attract the degradation of the RNA by Xrn1p/mXRN1p to ensure turnover
of an old RNA. mXRN1p itself does not contribute to G4
tetraplex formation because it does not catalyze the formation of this structure (Bashkirov, V.I., and W.-D.
Heyer, unpublished observation), unlike, for example, the
nuclear protein Rap1p of S. cerevisiae, which is involved
in telomere metabolism (Giraldo and Rhodes, 1994
). Alternatively, G4 RNA structures, which may be formed
quickly from short G-rich sequences (Kim et al., 1991
), may be used in the cell to squelch the activity of Xrn1p/
mXRN1p to achieve overall regulation of the exoribonuclease activity itself.
Implications of the mXRN1p Localization
A major distinction between eukaryotic and prokaryotic
organisms is the subcellular compartmentalization of the
eukaryotic cell in membrane-bound compartments including nucleus, cytoplasm, mitochondria, ER, and Golgi apparatus. In recent years this general organizational picture
of the eukaryotic cell was further refined when it was realized that molecular processes like RNA splicing (Fu and
Maniatis, 1990; for review see Spector, 1993
; Lamond and Carmo-Fonseca, 1993
) or DNA replication (Mills et al.,
1989
; for review see Spector, 1993
) are confined to subcompartments within the nucleus. Spatial isolation of lytic
activities is of advantage for the cell as demonstrated by
the existence of the lysosome compartment. Concentration and confinement of RNA hydrolytic activity in the cytosol is similarly advantageous to minimize a possible interference of the exonuclease with normal cellular processes. The mXRN1p-containing foci are possible sites of
RNA turnover in the cytoplasm. Alternatively, the
mXRN1p-containing foci may represent storage sites for
mXRN1p. However, this is unlikely because the cDNAs
do not encode an inactive precursor enzyme but rather an
active exoribonuclease (Fig. 7). It can be expected that not
only RNA turnover but also other molecular processes are
confined to specific sites in the eukaryotic cytosol. Because mXRN1p is the first RNA turnover protein to be localized in mammalian cells, colocalization studies with
other RNA turnover functions that would support the subcompartmentalization model are unfortunately not possible.
The majority of translatable mRNA in fibroblasts is associated with the cytoskeleton (Taneja et al., 1992). In developing Drosophila embryos and in nerve cells, localized
translation of specific mRNAs has been linked to cytoplasmic transport of mRNA along microtubules (for review
see St. Johnston, 1995), implying the presence of microtubule-associated proteins that link mRNA to transport along
microtubules. The codiscovery of the S. cerevisiae XRN1
gene as KEM1 (Kim et al., 1990
) has suggested that, in addition to being an exoribonuclease, the protein is a microtubule-associated protein (Interthal et al., 1995
). The majority of mouse mXRN1p is localized in foci, which do not
contain tubulin. However, the mXRN1p molecules localized more generally in the cytoplasm show general colocalization with the cytoplasmic tubulin network (Fig. 9 b).
Moreover, this general sublocalization of mXRN1p but
not the foci was abolished when the cells were incubated
with a microtubular inhibitor (Fig. 9 d). Cold treatment
mimicked this effect (Fig. 9 c), arguing against a drug artifact. This suggests that this subpopulation of mXRN1p
molecules is associated with microtubules, possibly relating RNA turnover to the cytoskeleton.
Received for publication 31 August 1996 and in revised form 27 November 1996.
Please address all correspondence to Wolf-Dietrich Heyer, Institute of General Microbiology, Baltzer-Str. 4, CH-3012 Bern, Switzerland. Tel.: 41 31 631 46 56. Fax: 41 31 631 46 84. e-mail: heyer{at}imb.unibe.chWe thank Drs. W. Filipowicz, J. Kohli, and Y. Nagamine for critically reading the manuscript, O. Bezzubova for kind help in the cDNA cloning, and Dr. R. Jessberger for providing the thymus library. Drs. P. Szankasi and G.R. Smith kindly communicated data before their publication. Drs. A. Johnson and R. Kolodner kindly supplied their overexpression vector. Drs. G. Giese and P. Traub kindly provided antivimentin antibody and mouse skin fibroblasts. Dr. H. Neitzel kindly provided the E10 cell line.
This work was supported by a career development award (Swiss Talents in Academic Research and Teaching) and a research grant of the Swiss National Science Foundation to W.-D. Heyer, and East-European collaborative grants of the Swiss National Science Foundation and an International Research Scholar's award from the Howard Hughes Medical Institute to V.I. Bashkirov and W.-D. Heyer. H. Scherthan was supported in part by the Deutsche Forschungsgemeinschaft. The Basel Institute of Immunology was founded and is supported by F. Hoffmann-La Roche & Co. Ltd.