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J Biol Chem, Vol. 274, Issue 9, 5925-5930, February 26, 1999
From the We have identified a cellular target for
proteasomal endonuclease activity. Thus, 20 S proteasomes interact with
the 3'-untranslated region of certain cytoplasmic mRNAs in
vivo, and 20 S proteasomes isolated from Friend leukemia
virus-infected mouse spleen cells were found to be associated with a
mRNA fragment showing great homology to the 3'-untranslated region
of tumor necrosis factor- During the last 2 years, the proteasome has emerged at the
forefront of modern cell biology. It is currently evident that the
proteasome plays a key role in multiple events. Thus, it is involved in
the regulation of transcription, antigen presentation, and the
degradation of damaged and misfolded proteins, and it drives the cell
cycle (1). In addition, there is evidence for the involvement of the
proteasome in post-transcriptional control of gene expression (2,
3).
The 20 S proteasome is a highly organized multimeric protein complex
forming a cylindrical structure that has been detected in all
eukaryotic cell systems investigated so far as well as in bacteria (1).
The proteasome cylinder consists of a stack of two In addition, proteasomes have been reported to contain about
0.0016-0.2% low molecular mass RNAs. The RNA content depends on the
origin of the proteasomes and decreases with increasing proteasome
purity (4, 5). These RNAs are heterogeneous in size but migrate most
frequently in a molecular size range of 70-200 nucleotides (5).
Partial sequence analysis of proteasomal RNAs has revealed that no
particular species of RNA is specifically associated with the
proteasomes. These proteasomal RNAs were suggested to represent
contaminations of purified proteasomes (4) or residual substrate
fragments of a specific proteasomal RNase activity (2). The latter
hypothesis is more attractive because it has been shown that
endonuclease activity is associated with subunits In the present paper, we provide evidence in vivo and
in vitro that 20 S proteasomes associate with 3'-UTR
mRNA fragments containing AUUUA motifs. In addition, we first
demonstrate that AUUUA-rich elements are degraded by 20 S
proteasome-associated RNase activity at specific cleavage sites.
Definition of TBK Buffers--
Buffer TBK X consists of 20 mM Tris-HCl, pH 7.4, x mM KCl, 2.5 mM MgCl2, and 7 mM
2-mercaptoethanol, where x is 100, 240, or 350.
Cell Fractionation Procedure and Isolation of
Proteasomes--
20 S proteasomes were extracted from Friend leukemia
virus-infected mouse spleen or calf liver by differential
centrifugations (9) and highly purified by fast protein liquid
chromatography (FPLC) as described previously (2).
Protein Gel Electrophoresis--
Proteins were precipitated with
10% trichloroacetic acid (final concentration) or 2 volumes of
ethanol. Electrophoresis of proteins was on one-dimensional
SDS-polyacrylamide gels (according to Ref. 10). Molecular mass markers
were: phosphorylase b (94 kDa), bovine serum albumin (68 kDa),
ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soybean trypsin
inhibitor (20 kDa), and lactalbumin (14 kDa).
Proteasome RNA Extraction and RNA
Electrophoresis--
Proteasomes from sucrose gradient fractions were
precipitated with 2 volumes of ethanol and sedimented by centrifugation
in a HB-4 rotor Sorvall (20 min, 9000 rpm, 4 °C). Pellets were
resuspended in Buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, pH 7.5, and 0.5% sodium
lauroylsarkosinat). Then 0.2 mg/ml of proteinase K was added, and
incubation was carried out for 1.5 h at 37 °C. RNA was
extracted from this suspension with chloroform/phenol (11) and
precipitated with 2.5 volumes ethanol and 0.3 M sodium acetate. Such purified RNA was labeled at the 3' end with cytidine 3'-5'-[5'-32P]biphosphate in a reaction catalyzed by T4
RNA ligase (12). Labeled proteasome RNA was separated on a 11%
sequencing gel as described previously (5).
Assay of Proteasome RNase Activity and RNA Sequencing--
The
quantity of proteasomes was determined by the Bio-Rad assay. The
synthetic oligomers were purchased from Eurogentec (Belgium). RNA
oligomers were labeled at the 5' end with [ [3H]Poly(U) Hybridization--
Identification of
poly(A)+ sequences in mRNAs was by hybridization with
[3H]poly(U) as described (14).
Cloning and Sequencing Analysis of the cDNAs of
Proteasome-associated RNAs--
This procedure was described in detail
(15).2 Briefly, proteasomes
RNA were extracted from proteasomes as described above and
polyadenylated (16). cDNAs were synthesized using oligo(dT) primers
as described (17). After synthesis of the DNA double strand and
ligation with a BglII linker, the proteasome DNA was inserted in a pGEM I plasmid and transformed into Escherichia coli DH1 strain (18). Finally, the plasmid was isolated and submitted to restriction endonuclease analysis (19). Plasmid DNA was
sequenced using the dideoxy procedure (20).
Detection of Proteasomes Associated with the 3' Ends of Cellular
mRNAs--
When a post-mitochondrial supernatant of mouse
erythroblasts was analyzed by sucrose gradient centrifugation,
proteasomes probed with a monoclonal antibody sedimented exclusively in
subribosomal fractions smaller than the ribosomal subunits 40 S (Fig.
1). In this sedimentation range,
proteasomes comigrated with a relative heterogeneous population of
poly(A)+ mRNAs, which was detected by
[3H]poly(U) hybridization (Fig. 1). As previously
reported (21), these mRNAs are not engaged in translation and
consist of a pool of senescent mRNAs and of mRNAs that are
stored for future translation. If proteasomes are involved in the
degradation of senescent mRNAs containing AREs, they should
interact with their 3'-UTR near the poly(A)+ tail. Moreover, if
the proteasome 20 S complex that is bound to 3'-UTR partially overlaps
with the poly(A)+ sequence, incubation with RNase A and RNase
T1 should release proteasome/UTR/poly(A)+ complexes because
poly(A)+ is not hydrolyzed under these conditions. To
investigate this idea more closely, the particles between 10 and 30 S
were pooled and analyzed by further centrifugation on 10-50% sucrose
gradients in Tris buffer containing 100 mM KCl (TBK 100).
These particles sedimented with two major maxima of absorbance in the
range of 13 S, a protein complex of unknown function we and others
observed earlier (5, 22), and 20 S, consisting mainly of proteasomes, and one smaller in the range of about 30 S (Fig.
2A). Poly(A)+
mRNAs sedimented between 10 and 30 S with major distribution in
zones corresponding to the maxima of absorbance (Fig. 2A). The presence of proteasomes was identified by their typical protein pattern (right panels of Fig. 2).
After incubation with RNase T1 and RNase A, [3H]poly(U)
hybridization revealed a sharp peak of labeled poly(A)+
containing particles in the range of 10 S (23, 24) and a smaller
population that sedimented in the range of 20 S, together with
proteasomes (Fig. 2B). However, when Rnase-treated particles were analyzed by centrifugation through sucrose gradients containing TBK 350, much less poly(A)+ containing fragments sedimented
with 20 S proteasomes (Fig. 2C). From these experiments, we
concluded that proteasomes retained poly(A)+ containing
mRNA fragments at 100 mM KCl, while they dissociated from the proteasome complex at 350 mM KCl. This is in good
agreement with results we have published earlier showing that
proteasomes dissociate completely from mRNAs at 350 mM
KCl (Refs. 5 and 7 and Fig.
3A). These observations
suggest that proteasomes interact directly with the poly(A)+
sequence or very nearby in the 3'-UTR of certain mRNAs.
Identification of a Proteasome-associated RNA Fragment with ARE
That Maps in the 3'-UTR of TNF
To elucidate this hypothesis, we sequenced some proteasome-associated
RNAs. For this approach, 10-30 S particles (Fig. 1) were pooled and
further sedimented through 10-50% sucrose gradients in TBK 350. Under
these conditions, poly(A)+ mRNAs dissociated completely
from 20 S proteasomes (Fig. 3A). Such purified proteasomes
were pooled (Fig. 3A, fractions 12 and 13), and the purity of the particles was tested by Laemmli
polyacrylamide gel electrophoresis. Fig. 3B shows the
typical pattern of proteasomal subunits migrating between 20 and 35 kDa
with some faint bands of nonproteasomal proteins in the range of 43-68 kDa.
Proteasome-associated RNAs were extracted from these fractions, labeled
at the 3' end with cytidine 3'-5'-[5'-32P]biphosphate,
and analyzed by RNA gel electrophoresis. Fig. 3C shows
typical profiles of proteasome-associated RNAs, which migrate as a
heterogeneous population ranging from 60 to 500 nucleotides. Highly
purified proteasomes washed with 1% lauroylsarkosyl contain almost one
RNA species, which was identified to be tRNALys3 (3, 25).
For sequencing, RNAs were extracted from less purified 20 S fractions.
After polyadenylation, cDNAs of proteasome-associated RNAs were
synthesized and cloned in a pGEM I transcription vector. Sequencing of
eight cDNA clones using dideoxynucleotides revealed quite different
sequences (15).2 We here show sequence number 4 (P4), which
contains several AUUUA motifs (Fig.
4A). Further computer analysis
revealed that this sequence is 86% homologous with the 3'-UTR of mouse
TNF Proteasomes Destabilize the 3'-UTR of TNF
In a first series of experiments, we incubated highly purified
proteasomes with [
To address this question, different synthetic oligoribonucleotides
containing no, two, or four copies of the AUUUA motif were labeled with
[ Despite the identification of several proteins that associate with
RNAs containing AUUUA motifs in vitro, none has been
definitively linked to ARE-mediated degradation of mRNAs. Most of
these proteins migrated on polyacrylamide gels in a range of 30-45
kDa. First identified was a protein with a molecular mass of 44 kDa
containing three subunits and named AUBF
(adenosine-uridine binding
factor (28). Using an in vitro destabilizing
system, Brewer (29) identified two polypeptides of 37 and 40 kDa that
are implicated in the acceleration of degradation of ARE containing
messengers. Others described a 32-kDa protein identical to HuR that
specifically cross-links to (AUUUA)4 and
(AUUUUA)3 but not (AUA)8 and
(AUUA)5 RNAs in vitro (30). This protein would
play an RNA-stabilizing role in the ARE-directed mRNA decay in
mammalian cells (31).
On the other hand, a cytoplasmic 20 S protein complex with unknown
protein composition was described to be involved in the destabilization
of AREs (26). This work was of particular interest because we have
recently shown that a similar cytoplasmic structure, the 20 S
proteasome, harbors a specific endonuclease activity (2). In addition,
the results in this paper clearly demonstrate that proteasomes
hydrolyze oligo-RNAs with (AUUUA)-rich multimers at specific sites
in vitro, which corresponds well with the in vivo
observations of other groups. Cellular mRNAs with ARE in their
3'-UTR are expressed transiently with half-lives in the order of 10-30
min (32), whereas The results suggest that proteasomes could inhibit the translation of
certain cytokine mRNAs and in general of other short-lived mRNAs using their ARE as target for degradation. Inappropriate translation of these cellular mRNAs has dramatic effects because their protein products interfere with cell division and cell
differentiation. Interestingly, viral mRNAs like tobacco mosaic
virus RNA with much less extended adenosine- and uridine-rich motifs
was reported to be a good substrate for proteasome-associated RNase
activity (2). Based on these observations, selective recognition of proteasomes RNA substrate and the association of proteasomes with RNA
sequences cannot be restricted to the presence of adenosine- and
uridine-rich elements alone. In fact, RNAs of different origin like
tRNALys3, viral RNAs, or 18 S rRNA that associate with
proteasomes suggest the existence of a common secondary structure or
box-like sequence. Indeed preliminary sequence comparison of these
mRNAs and RNA fragments we found in proteasomes preparations
revealed a common nucleotide motif that will be presented in a
subsequent paper. Taken together, we concluded that the endonuclease
activity associated with the We thank Dr. R. John Mayer for helpful discussions.
*
This work was supported in part by the European Community
Biomed II Programm, the Hasselblad Foundation, the Ministère de la Recherche et de la Technologie, the Conseil Régional
d'Auvergne, and the Fondation pour la Recherche Médicale
(Sidaction), France.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF109292.
¶
To whom correspondence should be addressed. Tel.:
33-4-73-40-51-03; Fax: 33-4-73-40-51-03; E-mail address:
hpschmid{at}cicsun.univbpclermont.fr.
2
F. Petit, A.-S. Jarrousse, C. Kreutzer-Schmid,
R. Gaedigk, and H.-P. Schmid, unpublished results.
The abbreviations used are:
UTR, untranslated
region;
ARE, adenosine- and uridine-rich element;
FPLC, fast protein
liquid chromatography;
TNF, tumor necrosis factor.
Possible Involvement of Proteasomes (Prosomes) in
AUUUA-mediated mRNA Decay*
,,,¶
Equipe "Protéasome et
Auto-Surveillance Cellulaire" OVGV UA INRA 987, Université
Blaise Pascal, Clermont-Ferrand II, 24 avenue des Landais 63177, Aubière cedex, France and the § Biologisches
Institut, University of Stuttgart, Pfaffenwaldring 47, D-70569 Stuttgart 80, Germany
ABSTRACT
Top
Abstract
Introduction
References
mRNA that contains AUUUA sequences. We
furthermore demonstrate that 20 S proteasomes destabilize
oligoribonucleotides corresponding to the 3'-untranslated region of
tumor necrosis factor-
, creating a specific cleavage pattern. The
cleavage reaction is accelerated with increasing number of AUUUA
motifs, and major cleavage sites are localized at the 5' side of the A
residues. These results strongly suggest that 20 S proteasomes could be
involved in the destabilization of cytokine mRNAs such as tumor
necrosis factor mRNAs and other short-lived mRNAs containing
AUUUA sequences.
INTRODUCTION
Top
Abstract
Introduction
References
discs and two
central rings, each of which is composed of seven subunits with
molecular masses between 19 and 35 kDa. Two-dimensional gel
electrophoresis revealed up to 20 individual proteins with the number
varying between cells and species. The rings of the 20 S proteasome
harbor at least five endopeptidase activities that are differently
regulated in an ATP-dependent fashion by two copies of a 19 S protein complex, which can associate with the discs of the 20 S
core proteasome, forming the 26 S proteasome with a molecular mass of
about 2000 kDa. This complex processes and degrades ubiquitinated
proteins (1).
and 
in the
discs of proteasomes, indicating the existence of a close and
specific relationship between proteasomes and RNA (6). In this context,
20 S proteasomes (prosomes) were detected as factors that control
translation of Friend leukemia virus-infected mouse erythroblasts by
transient association with free mRNAs (5, 7). How proteasomes
select senescent free mRNAs for degradation remains unknown. Work
on mRNA decay has shown that sequence elements such as the
3'-untranslated region (UTR)1
regulate the degradation of different eukaryotic messengers. The best
studied are the AUUUA-rich motifs within the 3'-UTR of short-lived
mRNAs named ARE. The mechanisms by which these mRNAs are
targeted for rapid degradation and the factors that are involved in
this association with ARE have not been definitively characterized (8).
EXPERIMENTAL PROCEDURES
-35S]ATP
(Amersham) or [-32P]ATP and T4 polynucleotide kinase
(Boehringer Mannheim) according to the manufacturer's instructions and
then incubated with 50 µg of proteasomes for 30 min at 37 °C in
TBK 240. The digests were extracted from enzymatic assays with
chloroform/phenol (11) and run on a 16% sequencing gel with 1× TBE
running buffer for 2 h at 50 watts. The ladder was obtained by
alkaline hydrolysis of RNA oligomers (13).
RESULTS
View larger version (32K):
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Fig. 1.
Sedimentation profile of the
post-polyribosomal supernatant. Pellets of post-polyribosomal
supernatants of Friend leukemia virus-infected spleen cells were
resuspended in TBK 100 and analyzed by centrifugation on 10-25% (w/w)
sucrose gradients (Beckmann rotor SW27, 20,000 rpm, 4 °C, 16 h). A, individual fractions (100 µl) were hybridized with
[3H]poly(U) to detect poly(A)+ containing
sequences. Solid line, absorbance at 254 nm; line
with filled circles, [3H]poly(U)
hybridization. B, fractions a-f were
precipitated with ethanol and analyzed by Laemmli polyacrylamide gel
electrophoresis. Proteins were transferred to nitrocellulose membranes,
and proteasomal antigens were probed by a monoclonal anti-pros P27
( ) antibody (IB5). a, fractions 1-4; b,
fractions 5-8; c, fractions 9-11; d, fractions
12-15; e, fractions 16-19; f, fractions 20 and
21; left lane, markers were FPLC purified proteasomes
analyzed by gel electrophoresis and immunoblotting (37).
View larger version (19K):
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Fig. 2.
Sedimentation profile of subribosomal
particles after RNase treatment. Particles of 12 gradients
sedimenting between 30 and 10 S (Fig. 1, fractions 10-18) were pooled
and concentrated by sedimentation (Beckmann rotor Ti60, 50,000 rpm,
4 °C, 19 h). Solid line, absorbance at 254 nm;
line with filled squares,
[3H]poly(U) hybridization (see Fig. 1); P,
proteasomes. Right panels, the protein pattern of the
proteasome. A, pellets were resuspended in TBK 100 and
analyzed by sedimentation through 10-50% (w/w) sucrose gradients
(Beckmann rotor SW40, 36,000 rpm, 4 °C, 18 h). B,
pellets resuspended in TBK 100 were incubated for 30 min at 37 °C
with RNase A (5 µg/ml) and RNase T1 (20 units/ml) and immediately
analyzed by sedimentation through 10-50% (w/w) sucrose gradients in
TBK 100 (Beckmann rotor SW40, 36,000 rpm, 4 °C, 18 h).
C, pellets resuspended in TBK 100 were incubated for 30 min
at 37 °C with RNase A (5 µg/ml) and RNase T1 (20 units/ml) and
immediately analyzed by sedimentation through 10-50% (w/w) sucrose
gradients in TBK 350 (Beckmann rotor SW40, 36,000 rpm, 4 °C, 18 h). D, schematic presentation of the RNase protection
experiments.
View larger version (20K):
[in a new window]
Fig. 3.
Sedimentation profile of subribosomal
particles and RNA content of proteasomes. A, particles
sedimenting between 30 and 10 S (Fig. 1, fractions 10-18) were pooled
and concentrated by sedimentation (Beckmann rotor Ti60, 50,000 rpm,
4 °C, 19 h). Pellets were resuspended in TBK 350 and analyzed
by sedimentation through 10-50% (w/w) sucrose gradients (Beckmann
rotor SW40, 36,000 rpm, 4 °C, 16 h). Solid line,
absorbance at 254 nm; line with filled circles,
[3H]poly(U) hybridization (see Fig. 1); P,
proteasomes. B, polyacrylamide gel electrophoresis analysis
of the proteasome purified by subsequent sucrose gradient
centrifugation (see panel A). Proteins were visualized by
Coomassie Blue staining. Lane 1, molecular mass markers
(kDa); lane 2, the protein profile of the proteasome.
C, proteasomes purified by FPLC or by subsequent sucrose
gradient centrifugation were treated with proteinase K, and proteasomal
RNAs were extracted with chloroform/phenol and analyzed on a 11%
sequencing gel. Lane 1, RNAs extracted from FPLC purified
proteasomes; lane 2, proteasomal RNAs extracted from
fractions 12 and 13 from sucrose gradients (see panel A);
lane 3, nucleotide markers: pBR322 digested with
MspI and labeled at the 5' end with
[ -32P]ATP.
mRNAs--
Another strong
argument for a possible association of proteasomes with mRNAs is
the presence of different smaller RNA molecules in proteasomes
preparations. The length of these RNAs and the RNA content varies from
preparation to preparation. Based on these findings, we postulated
recently that at least some of these RNA molecules could be residual
fragments of the RNA substrates cleaved by the endonuclease activity
that is associated with proteasomes (2, 3, 6).
mRNA (Fig. 4B).
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Fig. 4.
Comparison of proteasomal RNA P4 with
TNF mRNA. A, proteasomal
RNA P4 extracted from purified proteasomes as described previously
(Fig. 3C) was polyadenylated and corresponding cDNA was
synthesized, cloned in a pGEM I transcription vector, and sequenced.
The adenosine- and uridine-rich region is underlined.
B, computer analysis revealed high homology between P4 RNA
(top) and mouse TNF mRNA 3'-UTR (bottom).
Alignment was done with the help of the Fasta program using the
Infobiogen Molecular Biology Server. Inserted (-) and identical (:)
nucleotides are shown.
mRNA--
Several
cellular proteins acting as trans-factors were reported to
recognize AREs and may target, in some way, cytokine mRNAs like
TNF mRNA for degradation. Among these factors that associate specifically with ARE, a 20 S protein complex with unknown protein composition was described to be involved in the destabilization of ARE
(26). Because 20 S proteasomes and this 20 S protein complex share
common properties and because we have identified an RNA fragment
containing AUUUA motifs almost homologous to TNF mRNA in proteasome
preparations, we wanted to investigate whether proteasomes could be
involved in the degradation of ARE.
-35S]ATP-labeled 3'-UTR of murine
mRNA TNF that contains an extended adenosine- and uridine-rich
region. After extraction with chloroform/phenol, the digests were run
on a 16% polyacrylamide sequencing gel. Fig. 5A demonstrates that
proteasomes cleaved the TNF 3'-UTR in seven well defined fragments.
Comparison of the adenosine- and uridine-rich sequences of many
cytokine mRNAs including those that code for TNF led to the
identification of sequence motifs like AUUUA that are often present in
multiple copies. The TNF 3'-UTR we used for our experiments
contained five copies of this pentameric motif, four of which overlap
(AUUUAUUUA) (Fig. 5B). So we asked whether the number of
AUUUA motifs could modify the cleavage reaction.
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Fig. 5.
In vitro degradation of
TNF mRNA 3'-UTR. A,
7.105 cpm of murine TNF mRNA 3'-UTR labeled with
[-35S]ATP were incubated for 30 min at 37 °C in TBK
240 in the absence or presence of 50 µg of FPLC purified proteasomes
from calf liver cells. RNA digests were extracted from enzymatic assays
with chloroform/phenol and run on a 16% sequencing gel. The nucleotide
ladder was obtained by alkaline hydrolysis of TNF RNA. Lane
1, incubation of TNF mRNA 3'-UTR in the absence of
proteasomes; lane 2, incubation of TNF mRNA 3'-UTR in
the presence of proteasomes; lane 3, nucleotide ladder of
TNF mRNA 3'-UTR. B, TNF mRNA 3'-UTR sequence.
The cleavage sites (*) are indicated. The numbers denote the
nucleotides positions in the cDNAs of murine (Mu) TNF
(38).
-32P]ATP and used in degradation assays. We found
that a minimum of two AUUUA motifs is required for the destabilization
by proteasomes. We detected only one cleavage site with
(AUUUA)2 oligo-RNA, whereas incubation of
(AUUUA)4 oligo-RNA with proteasomes produced five fragments
corresponding to five cleavage sites (Fig.
6). Obviously, preferential cleavage
sites are situated at the 5' end of the nucleotide A within a AUUUA
motif. Moreover, the densitometric analysis of native oligonucleotides
containing two or four motifs after incubation with proteasomes for
various times revealed that (AUUUA)4 oligo-RNA was degraded
much faster than the oligo-RNA containing two AUUUA motifs (data not
shown). These data correlate well with in vivo experiments
(27).
View larger version (73K):
[in a new window]
Fig. 6.
In vitro degradation of RNA
oligomers containing multiple copies of AUUUA motifs.
A, (AUUUA)4, (AUUUA)2, and
(AUUUA)0 RNA oligomers labeled with
[ -32P]ATP were incubated in the absence or presence of
proteasomes as described in Fig. 5 and analyzed on a 16% sequencing
gel. Lane 1, (AUUUA)4 RNA oligomer incubated in
the absence of proteasomes; lane 2, (AUUUA)4 RNA
oligomer incubated in the presence of proteasomes; lane 3,
(AUUUA)4 nucleotide ladder; lane 4,
(AUUUA)2 RNA oligomer incubated in the presence of
proteasomes; lane 5, (AUUUA)2 RNA oligomer
incubated in the absence of proteasomes; lane 6,
(AUUUA)0 RNA oligomer incubated in the presence of
proteasomes. Full-length bands and sizes of the nucleotide ladder are
shown. B, RNA oligomers sequences. The cleavage sites (*)
are indicated.
DISCUSSION
-globin mRNA that contains only one AUUUA
motif is very stable with a half-life greater than 17 h (33).
Several studies have demonstrated that deletion or replacement of the
adenosine- and uridine-rich sequence in the 3'-UTR of c-fos
mRNA conferred significantly greater stability to the previously
unstable messenger (34), whereas the addition of a short DNA sequence
from the granulocyte-monocyte colony stimulating factor 3'-UTR into the
3'-UTR of the rabbit -globin gene destabilized the corresponding
naturally stable -globin mRNA (27). This could explain why
globin mRNA with one AUUUA motif was not degraded by
proteasome-associated endonuclease activity (2). For accelerated degradation, proteasomes apparently require multiple AUUUA motifs (at
least two) that partially overlap. Based on these results, we concluded
that proteasomes could be involved in the degradation of ARE. This
conclusion correlates well with the following observations: (i)
purified proteasomes from Friend leukemia virus-infected mouse spleen
cells contain adenosine- and uridine-rich RNA fragments with great
homology to the 3'-UTR of TNF mRNA (Fig. 4); (ii) Arrigo
et al. (35) have demonstrated by RNA sequencing that Drosophila proteasomes are associated with RNA fragments
that possess extended adenosine- and uridine-rich elements; and (iii) finally, our RNase protection experiments confirmed that proteasomes interact specifically with the 3' end of a nondefined number of cytoplasmic RNAs. However, the binding site is not the poly(A)+
sequence because proteasome-associated RNA fragments reported so far do
not have oligo(A) stretches, and proteasomes associate strongly with
TMV-RNA without poly(A)+ or with adenovirus mRNAs of which
the poly(A)+ tail is blocked by oligo(dT) (36). We suspect that
proteasomes bind to sequences adjacent or within adenosine- and
uridine-rich elements close to poly(A)+ and cleave the 3' end
with the poly(A)+ sequences of the corresponding messengers.
This idea fits well with the hypothesis of other groups that postulate
the existence of a specific cytoplasmic endonuclease activity to be
involved in post-transcriptional gene silencing (26).
subunits and of the 20 S
proteasome complex (6) is rather important for cellular events.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
Top
Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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 F De Conto, E Pilotti, S. Razin, F Ferraglia, G Geraud, C Arcangeletti, and K Scherrer In mouse myoblasts nuclear prosomes are associated with the nuclear matrix and accumulate preferentially in the perinucleolar areas J. Cell Sci., January 7, 2000; 113(13): 2399 - 2407. [Abstract] [PDF]
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J. S. Buzby, G. Brewer, and D. J. Nugent Developmental Regulation of RNA Transcript Destabilization by A + U-rich Elements is AUF1-dependent J. Biol. Chem., November 26, 1999; 274(48): 33973 - 33978. [Abstract] [Full Text] [PDF]
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W. S. Lai and P. J. Blackshear Interactions of CCCH Zinc Finger Proteins with mRNA. TRISTETRAPROLIN-MEDIATED AU-RICH ELEMENT-DEPENDENT mRNA DEGRADATION CAN OCCUR IN THE ABSENCE OF A POLY(A) TAIL J. Biol. Chem., June 15, 2001; 276(25): 23144 - 23154. [Abstract] [Full Text] [PDF]
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