University Hospital Freiburg, Department of Internal Medicine II, Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany
* Author for correspondence (e-mail: nassal2{at}ukl.uni-freiburg.de)
Accepted 6 November 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: E3 enzyme, Ubiquitin ligase, Ubiquitylation, RING finger
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A 3' terminally truncated 2.1 kb cDNA clone obtained by this
procedure was completed by fusion to an overlapping clone identified by
molecular hybridization. This assembled sequence contained a continuous large
open reading frame (ORF) for a 1208 amino acid protein with a calculated
molecular mass of 138 kDa. On the basis that it was isolated as an RNA-binding
protein and has ubiquitin-protein ligase (E3) activity (see below), it was
termed hRUL138 (human RNA-binding ubiquitin
ligase of 138 kDa). The relevance of the assembled clone
was independently confirmed by the publication, during the course of this
study, of a continuous cDNA in the HUGE (human unidentified gene-encoded large
proteins) collection (Kikuno et al.,
2000), KIAA0675 (Ishikawa et
al., 1998
), that encodes an identical protein. The corresponding
gene is located on chromosome 3 but the function of its predicted gene product
is unknown.
Database searches using the full-length sequence confirmed that hRUL138
represents a hitherto unknown human protein with no homologs in the completely
sequenced genomes of S. cerevisiae, C. elegan or A.
thaliana; however, highly related mouse cDNAs were recently identified
(S. G. Kreft, PhD thesis, University of Heidelberg, 2000). No known
RNA-binding motif (Perez-Canadillas and
Varani, 2001) was detectable. Motif searches indicated a central
coiled-coil domain and the potential presence of one to three transmembrane
regions; however, all had a weak score. The only really informative similarity
to other known proteins involved a short sequence stretch close to the C
terminus of hRUL138 that contains all of the essential Cys- and His-residues
of an intact RING (`really interesting new gene') domain
(Borden and Freemont, 1996
) of
the RING-H2 type [consensus:
CX2CX9-39CX1-3HX2-3H/CX2CX4-48CX2C;
C=Cys, H=His, X=any aa; H/C=His in RING-H2 and Cys in RING-HC fingers
(Borden, 2000
)]. The
recognizable features within the hRUL138 protein are schematically shown in
Fig. 1B (see Results for
further details).
|
RING domains are Zn2+-binding structures that were first
recognized for their ability to mediate protein-protein interactions
(Saurin et al., 1996).
Recently, they have been shown to be integral parts of a second major class,
in addition to HECT-domain proteins, of ubiquitin ligases (or E3 enzymes) in
which the RING is essential for function
(Borden, 2000
;
Freemont, 2000
;
Lorick et al., 1999
).
Ubiquitylation is a key event in proteasome-dependent protein degradation
(Pickart, 2000
;
Pickart and VanDemark, 2000
);
in addition it is important in many other processes that do not necessarily
involve proteolysis (Pickart,
2001
) such as signal transduction
(Wang et al., 2001
),
endocytosis (Hicke, 2001
) and
virus budding (Vogt, 2000
).
The mechanism of ubiquitylation (Pickart,
2001
) involves energy-dependent activation of the 76 amino acid
ubiquitin protein with an ubiquitin-activating (or E1) enzyme and its transfer
to a ubiquitin-conjugating (or E2) enzyme. Covalent ubiquitin attachment to
the
-amino group of lysines, or sometimes to the N terminus, of target
proteins is mediated by E3 enzymes; further ubiquitins are usually, but not
always, attached to the first one, leading to polyubiquitylation. E3s are the
main determinant for target specificity; consistently, there is a greater
variety of E3 than E2 enzymes, whereas only one E1 is known in most eukaryotes
including humans.
Together these considerations suggested that hRUL138 might be a novel RING-H2 E3 that can bind RNA. The functional analysis of hRUL138 reported here fully supports this notion.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid constructs
A full-length hRUL138 cDNA was assembled from the cDNA fragments contained
in plasmids pBlsNIII and ICRFp512H14173Q2 using an AvrII restriction
site located in the overlap region (nt position 1704 of the hRUL138 ORF). Both
fragments were cloned into the vector pT7AMVpol16
(Weber et al., 1994). The
resulting plasmid, pT7-hRUL138, comprises the complete hRUL138 ORF under
control of the bacteriophage T7 promoter. It also served as starting material
for various hRUL138 derivatives. For expression in E. coli, hRUL138
or wildtype and mutant fragments thereof were fused to the maltose-binding
protein (MBP) using the pMal-c2 vector (New England Biolabs); His-tagged
variants were obtained using the pET-30a(+) vector (Novagen). In some
constructs a FLAG epitope sequence (DYKDDDDK) was inserted using
oligonucleotide-mediated mutagenesis. Point mutations in the Lys-rich motif
(amino acids 662 to 666 KKKTK changed to SGSTA) and in the RING-H2 domain
(Cys1187 changed to Ser, i.e. C1187S) were generated by PCR-mediated
mutagenesis. Eukaryotic hRUL138 expression vectors were based on plasmid
pTR-UF5 (Zolotukhin et al.,
1996
), which encodes a codon-optimized version of the enhanced
green fluorescent protein (eGFP) under control of the CMV-IE enhancer/promoter
and an SV40 intron plus poly-adenylation signal. Appropriate hRUL138-encoding
fragments were inserted so as to generate C-terminal fusions with eGFP.
All plasmids were named according to the following scheme: the first letters indicate the parental plasmid (pT7, pMBP, pET, pTR-UF); the numbers following the term RUL give the amino-acid positions of the full-length hRUL138 present in the construct; amino acid substitutions are indicated in parentheses and the presence of tags by an acronym either in front of (N-terminal tags) or after (C-terminal tags) the RUL term. For instance, pMBP-RUL681-1128-FLAG stands for a plasmid encoding hRUL138 amino acids 681 to 1128 with a N-terminal MBP and a C terminal FLAG tag; the same names without the plasmid-specific term denote the corresponding proteins. As a marker for the endoplasmic reticulum (ER), the dsRed2 gene from plasmid pdsRed2 (Clontech) was used to replace the eYFP gene in pEYFP-ER (Clontech), thus maintaining the original calreticulin ER-targeting and KDEL ER retrieval signals. The corresponding plasmid was named pRFP2-ER. Detailed outlines of all cloning procedures are available from the authors upon request.
In vitro translation
In vitro translations were performed in rabbit reticulocyte lysate using
the TNT T7 Quick Coupled Transcription/Translation system (Promega) programmed
with appropriately linearized pT7 plasmids. For 35S-labeling,
[35S]methionine (specific activity 1,000 Ci/mmol;
Amersham/Pharmacia) was used at 25 µCi per 50 µl reaction.
Expression of recombinant proteins
MBP fusion proteins were expressed in E. coli strain Top10
(Invitrogen), pET constructs in E. coli BL21(DE3) cells (Novagen).
MBP-tagged proteins were enriched by using amylose resin (New England Biolabs)
and His-tagged variants by using Ni2+ nitrilotriacetate (Ni-NTA)
agarose (Qiagen). For co-expression, BL21(DE3) cells were double-transformed
with ampicillin-resistance-mediating pMBP and kanamycin-resistance-mediating
pET vectors and propagated in the presence of both antibiotics. Protein
concentrations were estimated by comparing, on Coomassie blue stained SDS-PAGE
gels, the corresponding band intensities with those of serial dilutions of a
bovine serum albumin (BSA) standard of known concentration.
Cell culture and DNA transfection
Huh7 human hepatoma cells were maintained in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics
(Nassal, 1992). DNA
transfections were carried out using FuGENE 6 transfection reagent (Roche) as
suggested by the manufacturer.
Northern blot analysis
A human multiple tissue Northern blot (Clontech) was hybridized with a
[32P]dATP random primed probe (High Prime DNA labeling kit, Roche)
corresponding to nt 1497-1998 of the hRUL138 ORF or a full-length actin
control probe (Clontech) using standard conditions; hybridizing bands were
visualized on a BAS-1500 Phosphoimager (Fuji).
Western blot analyses
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)-resolved proteins were transferred onto PVDF membranes
(Amersham/Pharmacia) and incubated with the appropriate primary antibody
followed by horseradish-peroxidase-conjugated secondary antibodies (Dianova)
as described previously (König et
al., 1998). Immunoreactive bands were visualized using the
ECL-plus system (Amersham/Pharmacia). The following antibodies were used:
anti-FLAG monoclonal antibody M2 (Sigma); anti-His monoclonal antibody
anti-Tetra-His (Qiagen) and a polyclonal rabbit antiserum recognizing the
His-tag-containing linker sequence encoded by the pET-30 vector.
RNA-binding assays
DIG-labeled RNA probes were obtained by in vitro transcription (DIG RNA
labeling kit; Roche) of an appropriately linearized T7 promoter containing
plasmids as suggested by the manufacturer. The parental plasmid for the HBV-RT
and AMV RNA probes was pT7HAMV-RT (provided by J. Beck) which contains HBV
nucleotides 938-1130, that is, part of the reverse transcriptase ORF, behind
the 49 nt AMV leader. The HBV-RT probe consisted of both segments, the AMV
probe of only the AMV part. Labeled RNA was purified by gel filtration (G-25
Sephadex Quick Spin columns, Roche) and quantified via dot blotting (DIG
Nucleic Acid detection Kit, Roche). 32P-labeled RNAs were obtained
by in vitro transcription in the presence of 32P-CTP (specific
activity: 800 Ci/mmol; Amersham/Pharmacia) and quantified by Cerenkov
scintillation counting.
Northwestern blotting using DIG-labeled RNA probes was performed
essentially as described previously
(Gatignol et al., 1991).
Briefly, crude bacterial lysate (equivalent to 75 µl bacterial culture) or
partially purified bacterially expressed proteins (100 ng to 2 µg) were
resolved by SDS-PAGE and blotted onto a PVDF membrane. Membranes were
sequentially immersed, at room temperature, in binding buffer 1 (BB1; 50 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) containing
decreasing concentrations of guanidine-HCl (5 minutes each in 6 M, 3 M, 1.5 M,
0.75 M, 0.37 M and 0.18 M). Thereafter the membranes were rinsed in BB1,
blocked for 1 hour in 2.5% skim milk powder in BB1 and rinsed twice in BB1; in
some experiments an additional wash with 1 mg/ml of heparin in BB1 was
included. RNA binding was carried out in BB1 containing 50-100 ng/ml of the
corresponding DIG-labeled RNA probe and 10 µg/ml each of yeast RNA and
herring sperm DNA for 1 hour at room temperature. After three washes in BB1,
bound DIG-labeled RNA was detected via chemiluminescence (DIG Nucleic Acid
detection kit; Roche) on X-ray film.
For in-solution RNA-binding assays, approximately 2 µg (about 40 pmol;
400 nM) of the corresponding bacterially expressed His-tagged hRUL138
fragment, enriched by Ni-NTA agarose chromatography and dialysed against 20 mM
Tris-HCl (pH 7.5), was incubated with 2x106 cpm
HBV--containing RNA [6x107 cpm/pmol; 40 fmol (300 pM)
in binding buffer 2 (BB2; 50 mM Tris-HCl pH 7.5, 350 mM NaCl, 10 mM
ß-mercaptoethanol, 10 µg/ml yeast tRNA)] containing 0.4 U/µl RNasin
(Promega) in a total volume of 100 µl for 1 hour at 30°C. RNP complexes
were separated from free RNA by adding to the samples 40 µl (gel bed)
Ni-NTA agarose in 400 µl BB2 containing 20 mM imidazol (pH 7.9), followed
by a 30 minute incubation at 4°C with gentle agitation. After four washes
with 1 ml each of cold BB2 containing 20 mM imidazol-bound
32P-labeled RNAs were quantified by Cerenkov counting.
In vitro ubiquitylation assays
For self-ubiquitylation assays, hRUL138 and its variants were in vitro
translated in the presence of [35S]methionine. 2 µl of the
reaction mix was then incubated in a total volume of 50 µl of
ubiquitylation buffer (UB; 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM
dithiothreitol, 5.6 mM MgCl2, 4 mM ATP) with 4 µg ubiquitin
(Sigma) or methylated ubiquitin (Calbiochem) and approximately 10 ng of E1 and
100 ng of the respective E2 (all kindly provided by Martin Scheffner). As
E1-purified His-tagged wheat germ E1 expressed in insect cells was used,
lysates from E. coli cells overexpressing either UbcH5 or UbcH7
served as a source for E2 (Nuber et al.,
1996). For trans-ubiquitylation assays E. coli lysates
containing co-expressed MBP-RUL681-1208-FLAG and His-RUL681-1128-His were
passed through amylose resin and the proteins bound to 6 µl of amylose gel
were incubated in ubiquitylation buffer as described above, except that 10
µg of methylated ubiquitin were used. His-RUL681-1128-His-only
ubiquitylation reactions were performed with crude bacterial lysates.
Reactions were allowed to proceed for 90 minutes at 30°C and were
terminated by boiling for 5 minutes in SDS sample buffer. Samples were
analyzed by SDS-PAGE and autoradiography (when 35S labeled) or by
western blotting.
Proteasome inhibition
Huh7 cells were transfected with expression constructs for GFP- or
FLAG-tagged hRUL138 derivatives. 62 hours post-transfection, the cells were
incubated with a final concentration of 50 µM MG132 (Calbiochem) in DMSO
for 6 hours. Thereafter, they were washed twice with PBS, scraped off the
plate and lysed in SDS sample buffer. Equal aliquots from the lysates were
analyzed by western blotting using GFP- or FLAG-specific antibodies. Lysates
from nontransfected untreated cells and from cells incubated with the same
concentration of DMSO only served as controls.
Immunofluorescence
Transfected Huh7 cells were grown on glass coverslips. 48 hours
post-transfection GFP and/or RFP derivatives were examined directly with a
confocal laser scanning microscope (Zeiss LSM410). For nuclear
counter-staining with TOTO-3 iodide (Molecular Probes) the cells were fixed
with 2% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized for
10 minutes using 0.05% saponin in PBS and incubated for 1 hour with a mix of
two monoclonal anti-GFP antibodies (Roche) at a 1:1000 dilution in PBS
containing 3% bovine serum albumin and 0.05% saponin. After washing and
incubation with Alexa-488-conjugated goat-anti-mouse IgG (Molecular Probes)
for 1 hour, the cells were incubated with PBS containing 200 µg/ml of RNase
A for 30 minutes, washed with PBS containing 0.05% saponin for 10 minutes and
incubated with 1 µM TOTO-3 iodide in PBS. After rinsing with PBS, the
coverslips were mounted with Vectashield H-1000 (Vector Laboratories). Image
files were processed using Adobe Photoshop 5.5 software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A tBLASTn search (NCBI; URL:
http://www.ncbi.nlm.nih.gov:80/BLAST/)
with the full-length hRUL138 protein sequence in the H. sapiens
translated RNA database showed, besides the KIAA0675 product, only two
hypothetical ORFs with extended similarity, corresponding to amino acids
128-339 and the region from amino acid 740 to near the C-terminus. One is the
predicted tetratricopeptide repeat protein 3 (or TPR protein D, TPRD; GenBank
Accession Number D83077) on chromosome 21
(Ohira et al., 1996), the
other a similar, hypothetical gene on the X chromosome (GenBank Accession
Number XM_032867). The function of neither of the two proteins is known, and
the region of similarity between TPRD and hRUL138 does not involve the four
TPR repeats (Small and Peeters,
2000
) that gave TPRD its name. The central region of hRUL138,
approximately from amino acids 600 to 950, showed some similarity to a variety
of proteins containing myosin heavy chain tail-like potential coiled-coil
sequences (Lupas, 1996
); the
coiled-coil prediction is strongest for the sequence between position 794 to
852, extending with weaker scores in both directions from there (T. Doerks and
P. Bork, personal communication). Different search programs for transmembrane
regions (PHDhtm, SOSUI, TMAP, TMHMM, TMpred) predicted one, two or three
transmembrane helices around amino acids 120 to 140, 390 to 410, and 1001 to
1019; however, all had a low score that varied from program to program.
Although these sequence analyses did not offer any distinct clues to the
potential function of hRUL138, the C-terminal RING-H2 domain contained all of
the absolutely conserved Cys- and His-residues that are typical for this
motif, which occurs as a module in various proteins of otherwise unrelated
primary sequence (Borden,
2000); functionally, however, there is accumulating evidence that
most RING finger proteins may be protein-ubiquitin ligases
(Freemont, 2000
). We therefore
went on to experimentally address the RNA-binding potential, ubiquitin ligase
activity, expression profile and intracellular localization of hRUL138.
A lysine-rich region in the center of hRUL138 is critical for RNA
binding
The hRUL138 sequence did not contain a known RNA-binding motif; however,
because the truncated protein encoded by the initial cDNA had been identified
via its RNA-binding ability, the region responsible had to be present within
the first 666 amino acids. Deletion mapping combined with Northwestern
blotting was used to further define this region. A candidate was a stretch of
basic residues (amino acid positions 656 to 674, with 11 Lys plus 1 Arg
residue within a total of 19 amino acids; see
Fig. 2) that remotely resembled
the Arg-rich RNA-binding part within the HIV-1 Tat protein
(Cheng et al., 2001;
Smith et al., 2000
). In a
first series of experiments hRUL138 fragments corresponding to residues 1 to
205, 1 to 499 and 1 to 666 were expressed in E. coli as
maltose-binding protein (MBP) fusions. Crude bacterial lysates were subjected
to Northwestern blotting with the HBV-
-containing RNA probe in the
presence of 10 µg/ml of yeast RNA and herring sperm DNA as nonspecific
competitors. Specific signals were obtained only with longest protein (data
not shown). As this included, at its C-terminus, part of the suspected basic
region, three variants lacking 3, 6 or 9 amino acids from the C-terminus were
analysed in parallel to the 1 to 666 derivative
(Fig. 2A). Again, only the
latter generated signals in the Northwestern blot, one at the 120 kDa position
expected for the intact fusion protein and a second band at about 50 kDa, most
probably a cleavage product lacking the MBP part. Total protein loading on all
lanes was similar, as shown by Coomassie blue staining of the gel used to
generate the blot (right panel in Fig.
2A); this also excluded the possibility that the signals arose
from RNA binding to E. coli proteins. Hence amino acids 664 to 666
(KTK) are important for RNA binding.
|
RNA binding was independent of the C-terminal disposition of the basic motif and of the fusion to MBP, as shown by the variant K4-. In this pET30-vector-derived His-tagged construct, the four central Lys residues between positions 662 to 666 were replaced by neutral amino acids (KKKTK>SGSTA) in the context of hRUL138 amino acids 510 to 878; hence it still contained the rest of the Lys-rich motif including four further lysines, that is, the total number of basic residues was maintained. The corresponding wild-type construct, and an additional one coding for a C-proximal hRUL138 fragment of similar size (amino acids 832 to 1176) served as controls. The E. coli-expressed proteins were enriched by Ni-NTA agarose chromatography and analyzed by Northwestern blotting (Fig. 2B, left panel). No signals were observed for the C-proximal fragment and for the mutated 510 to 878 protein; by contrast, a series of bands was generated with the wild-type fragment 510-878; the largest was present at the 50 kDa position expected for the intact fusion protein. Reprobing the same blot with an antibody against the N-terminal His-tag (Fig. 2B, right) revealed similar amounts of the two 510 to 878 proteins plus several smaller, probably proteolytically derived products of similar mobility to the faster migrating bands on the Northwestern blot. The nonbinding C-proximal fragment was present in much larger amounts, indicating that the Northwestern signals did not originate from unspecific binding of the probe. In some experiments, and upon long exposure of the autoradiograms, weak signals were also observed with the K4- mutant 510 to 878 protein. Hence mutating the four central Lys-residues in the Lys-rich motif led to a drastic reduction, though possibly not complete abolishment, of RNA binding.
This was further corroborated by in-solution RNA-binding assays. The wild-type and the mutated 510 to 878 fragments were expressed as fusions with the pET30 linker; an N-proximal hRUL138 fragment (amino acids 1 to 205) fused in the same way served as a control. The proteins were incubated with 32P-labeled HBV RNA in the presence of 10 µg/ml of yeast tRNA and 350 mM NaCl, bound to Ni-NTA agarose, and after several washing steps the radioactivity remaining on the beads was determined by liquid scintillation counting; the presence of similar amounts of immobilized protein was confirmed by SDS-PAGE (data not shown). Compared to the wild-type protein, about 10-fold less RNA was retained by the K4- variant and about 20-fold less by the 1-205 fragment (Fig. 2C). These data confirmed that, also in solution, the Lys-rich motif is crucially involved in RNA binding.
In order to test whether hRUL138 binds only to HBV--containing RNA,
similar Northwestern experiments were performed using other RNA probes. These
included sequences from another part of the HBV genome (HBV-RT, without the
stem-loop) and an A-U-rich unstructured RNA derived from the alfalfa
mosaic virus (AMV) leader (Weber et al.,
1994
). On Northwestern blots loaded with the same wild-type and
K4- mutant 510-878 proteins as in
Fig. 2, all RNAs gave signals,
with the wild-type protein, of similar intensity to the original probe
(Fig. 3). These data did
therefore not reveal a significant preference of hRUL138 for certain RNA
sequences or structures. Additional in-solution experiments (data not shown),
in which RNA homopolymers were used as competitors, showed that poly-A and
poly-U, at 10 µg/ml, reduced binding of the labeled HBV
probe to the
immobilized wild-type 510 to 878 fragment by about 80%, whereas 100 µg/ml
of poly-G and poly-C were required to achieve a similar reduction;
double-stranded poly-IC was ineffective even at this concentration. These data
showed that hRUL does not indiscriminately bind to all RNAs but, because the
in vitro assays may not faithfully mimic physiological conditions, it remains
an open question whether RNA binding specificity is naturally broad or whether
specific natural targets exist that we currently do not know.
|
The hRUL138 RING-H2 domain is functionally active in self- and
trans-ubiquitylation
Many, if not all, RING proteins may be ubiquitin ligases, with an intact
RING being essential for ubiquitylation activity. E3 activity can be
reconstituted in vitro by providing to an E3 enzyme ubiquitin, an
ubiquitin-activating E1 and a conjugating E2 enzyme plus ATP and the target
protein; successful reaction results in an increased molecular weight caused
by polyubiquitylation. In the absence of a trans-target, various E3s have been
shown to self-ubiquitylate (Bays et al.,
2001; Fang et al.,
2000
; Lorick et al.,
1999
; Nuber et al.,
1998
). As a first test for E3 activity we investigated whether
hRUL138 is capable of self-ubiquitylation. The protein was in vitro translated
in rabbit reticulocyte lysate (RRL) in the presence of 35S-Met and
incubated with a reaction mix containing ATP, ubiquitin and recombinant E1
plus either UbcH5 or UbcH7 as the E2. In control reactions, individual
components were omitted. Incubation with the complete mix containing UbcH5
dramatically decreased the mobility of hRUL138, preventing most of it from
entering the stacking gel (Fig.
4A, lane 4); only a small proportion remained at the original
position. UbcH7, by contrast, had only minor, if any, effects. In the UbcH5
reaction without added ubiquitin, some decrease in the intensity of the
unmodified hRUL138 band and a concomitant accumulation of labeled material at
the border between stacking and separating gel were observed
(Fig. 4A, lane 6), most
probably due to the endogenous ubiquitylation components known to be present
in RRL (Mastrandrea et al.,
1999
). To prove that the observed mobility shift was caused by
polyubiquitylation, similar assays were performed using methylated ubiquitin
[MeUb (Hershko and Heller,
1985
)]; chemical blocking of the lysine
-amino groups
prevents formation of polyubiquitin chains (but not multiple
mono-ubiquitylation of a target containing more than one modifiable lysine
residue). Again, a marked mobility shift was induced, but this time,
expectedly, the products did not migrate as high up in the gel
(Fig. 4B, compare lanes 9 and
10). The efficiency of modification and the differences between ubiquitin and
MeUb were even more obvious when a C-terminal RING-containing fragment,
RUL681-1208, was used (Fig.
4B). Hence the observed mobility shifts are due to
auto-ubiquitylation of hRUL138.
|
To prove that an intact RING-domain was required for auto-ubiquitylation
the last of the essential Cys-residues in the RING motif was replaced by Ser
(variant C1187S); a corresponding mutation in the established RING-E3 ligase
Hrd1p abolished its activity (Bays et al.,
2001). No reaction was detected using the hRUL138 mutant whereas,
in a parallel assay with the wild-type protein
(Fig. 4C, lane 3), the
above-described results were fully reproduced. These data confirmed that
hRUL138 is capable of E2- and RING-dependent auto-ubiquitylation.
A more rigorous proof of genuine E3 activity is the ability to mediate
ubiquitylation of a target protein in trans. During purification of
bacterially expressed MBP derivatives of hRUL138 we had repeatedly observed
that proteolytic fragments lacking the MBP part co-eluted with the full-length
protein from the amylose affinity matrix. This suggested that hRUL138 was able
to dimerize, or oligomerize. Such a homomeric interaction would allow one to
bring a ubiquitylation-deficient hRUL138 variant lacking the RING-H2 motif in
close proximity to a self-ubiquitylation-competent hRUL138 molecule,
potentially leading to trans-ubiquitylation. Because full-length hRUL138 was
poorly expressed in bacteria, RUL681-1208, known to be active after in vitro
translation (Fig. 4B), was
fused with an N-terminal MBP and a C-terminal FLAG tag (MBP-RUL681-1208-FLAG)
and co-expressed, in E. coli, with a His-tagged fragment lacking the
RING-H2 motif (His-RUL681-1128-His). Binding of both proteins to the amylose
resin was confirmed by western blotting (data not shown). The immobilized
complexes were then subjected to ubiquitylation assays with MeUb. The reaction
products were separated by SDS-PAGE and visualized by western blots using
anti-His (for the trans-target) or anti-FLAG antibodies. The anti-His blot at
0 minutes incubation time showed the expected 58 kDa product plus a second
anti-His reactive band slightly above the 97 kDa marker, possibly a dimer.
Irrespective of the exact identity of this additional product (which was
observed only in the co-expression experiments), it was evident that upon
incubation with the ubiquitylation-proficient derivative
(Fig. 5, -His, lane 2),
but not in its absence (lane 6), a series of new products with lower mobility
was generated at the cost of the unmodified proteins. Interestingly, only a
weak reaction was observed when both proteins were separately expressed and
then mixed (lane 8); we assume this difference is mainly caused by a more
efficient hetero-oligomerization when the two proteins are co-expressed,
whereas subunit exchange between separately expressed homo-oligomers is slow.
Anti-FLAG blots (Fig. 5,
-FLAG) confirmed that MBP-RUL681-1208-FLAG expressed in E.
coli was competent for auto-ubiquitylation. These data demonstrated that
hRUL138 is able to mediate trans-ubiquitylation and therefore has genuine E3
ligase activity. We note that these experiments were performed with RUL
fragments lacking the Lys-rich region; RNA binding was therefore not required
for the auto-ubiquitylation and the special setting of the
trans-ubiquitylation assays.
|
The RING-H2 domain mediates proteasome-dependent degradation of
hRUL138 in vivo
To address the potential physiological relevance of the in vitro
auto-ubiquitylation reaction, we examined the influence on steady-state levels
of hRUL138 in transfected Huh7 cells of the established inhibitor of
proteasome-dependent protein degradation, MG132
(Palombella et al., 1994;
Rock et al., 1994
). For
detection by western blotting (and for fluorescence microscopy; see below) the
full-length hRUL138 protein and the RING-deleted hRUL1-1128 variant were fused
to GFP, against which highly specific antibodies are available. Because the
inhibitor is used dissolved in DMSO, control cells were treated with solvent
alone. Aliquots from SDS lysates of MG132 and DMSO-only treated cells
containing equal amounts of total protein were then analyzed by using a
monoclonal anti-GFP antibody (Fig.
6). For the full-length fusion protein, the signals at the
expected position were markedly enhanced by the inhibitor; the RING-deleted
fusion, by contrast, produced much stronger signals without the inhibitor, and
the signals remained unchanged in its presence. To exclude the possibility
that GFP mediated the effects, hRUL138 was, in addition, fused to the FLAG
peptide instead of GFP and analyzed correspondingly using an anti-FLAG
antibody (Fig. 6, right panel).
Again, the presence of MG132 led to a strong signal increase; specificity of
detection was confirmed by the absence of signals at the relevant position in
non-transfected MG132-treated Huh7 cells. Therefore, the RING-H2 domain in
hRUL138-mediated in vitro auto-ubiquitylation is also involved in regulating,
in a proteasome-dependent fashion, the steady-state levels of hRUL138 in
intact cells. The stabilizing effect of deleting the RING-H2 domain was also
observed when the GFP fusions were analyzed by fluorescence microscopy (see
below).
|
The rul138 gene is expressed at low levels in most tissues
and slightly higher in skeletal muscle, heart and kidney
To analyze the tissue specificity of hRUL138 RNA expression, premade
Northern blots containing polyA+ RNA from eight different tissues
were hybridized with the above-described 502 bp random-primed hRUL138 cDNA
probe. A 2 day exposure on the phosphoimager was required to generate the
autoradiogram shown in Fig. 7.
This revealed, in all tissues tested, a specific band at a position of about
4.8 kb; its intensity was highest in skeletal muscle and decreased from heart
and kidney, over the brain and pancreas to the lung, liver and placenta. In
addition, weaker bands at about 7.5 kb and, still weaker, at about 5.5 kb were
detectable in the tissues with the highest levels of the 4.8 kb transcript. In
the other tissues, the presence of these larger products could not
unambiguously be demonstrated. The 4.8 kb mRNA is large enough to accommodate
the entire hRUL138 ORF plus untranslated 5' and 3' regions. The
other bands probably represent alternative splicing products. To control for
equal RNA loading on the individual lanes, and for a semiquantitative
comparison, the same blot was reprobed using a ß-actin probe of similar
specific activity specific. After a 3 hour exposure, strong bands were visible
at the 2.0 kb position expected for ß-actin, and in addition at 1.8 kb
for -actin in heart and muscle. Although the weak hRUL138-specific
bands precluded more accurate quantifications we estimate that they were at
least ten times, and probably hundred times, less abundant than ß-actin
mRNA. Hence hRUL138 mRNA is expressed at low levels. Direct confirmation of
these data on the protein level will have to await the availability of
specific, high-affinity antibodies to hRUL138.
|
hRUL138 is excluded from nucleus and associated with a structured
cytoplasmic compartment
To investigate the intracellular distribution of hRUL138, plasmids encoding
the above-described GFP fusions plus several additional derivatives were
transiently transfected into Huh7 human hepatoma cells. Confocal laser
scanning fluorescence microscopy showed, for the full-length fusion protein, a
weak fluorescence concentrated in some cytoplasmic structures but excluded
from the nucleus (Fig. 8A).
This was seen in various experiments but always only very few cells per dish
exhibited clearly visible signals, suggesting that the full-length protein was
poorly expressed, or unstable, or its expression led to a preferential loss of
transfected cells. On the basis of the increased steady-state levels of
full-length hRUL upon proteasome inhibition demonstrated by western blotting
(see above) we incubated the transfected cells for 6 hours with MG132. This
increased the fraction of detectably positive cells, as monitored by visual
inspection; however fluorescence intensities were still very low.
Substantially more and stronger fluorescent cells were reproducibly observed
with a C-terminally truncated construct lacking the RING domain
(RUL1-1128-GFP), consistent with the anti-GFP western blot data shown above
(Fig. 6). Its intracellular
distribution appeared very similar to that of the full-length fusion protein
(Fig. 8B), indicating that the
RING domain is not responsible for the distinct localization. Exclusion from
the nucleus was confirmed by nuclear counterstaining using TOTO-3 iodide
(Fig. 8B, right panel); hence
the K-rich motif, despite some sequence similarity, is not active as a nuclear
localization signal. Suspecting that the compartment in question might be the
ER, we generated a variant of an improved version of the red fluorescent
protein with ER targeting and retention signals (RFP2-ER) on the basis of the
commercially available yellow fluorescent protein (YFP)-ER derivative
(Clontech). Huh7 cells transfected with this construct showed cytoplasmic
structures, sometimes accumulating around the nuclei, that were
indistinguishable from those stained with the YFP-ER variant (data not shown).
Overall the fluorescence distribution appeared very similar to that observed
with the hRUL138 derivatives. For confirmation we sought to demonstrate
colocalization of the proteins by co-transfection. The patterns looked very
similar upon separate inspection of the green and red channels and regions of
apparent co-staining were revealed (Fig.
8C). However, there were always regions with preferential red or
green staining; hence the localization of the two proteins was not completely
overlapping. Such a partial but incomplete overlap was observed in numerous
experiments. We also noted that in some cells, and increasing with time in
culture, the hRUL138-derived fluorescence appeared to accumulate in the
nuclear periphery, whereas the RFP2-ER signals remained more dispersed in the
cytoplasm, suggesting a segregation of the two proteins. Preliminary
experiments aimed at defining the localization signal(s) within the RUL138
sequence using variously truncated derivatives showed similar patterns even
for a variant consisting of only amino acids 510 to 878
(Fig. 8D). This suggests that
none of the putative transmembrane regions is crucial for association with the
compartment; possibly, association is mediated by the coiled-coil region in
hRUL138 but the exact nature of that compartment as well as the localization
mechanism remains to be determined.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA binding by hRUL138
In the absence of a known RNA recognition motif within the hRUL138 sequence
we used deletion mapping and Northwestern blotting to identify a central,
basic region (amino acids 656 to 674) as important for RNA binding. The first
seven lysines, present in the product of the initial cDNA clone, but not the
first five, were sufficient for RNA binding. Binding was also detected when
the basic motif was disposed internally rather than at the C-terminus and was
drastically reduced, though not completely abolished, when the central KKKTK
sequence was mutated to SGSTA; this was also observed in solution assays.
Because of the poly-cationic nature of the K-rich motif it might be argued
that the RNA binding detected in these assays is merely electrostatic.
However, several lines of evidence argue strongly against this view. First,
all assays were performed in the presence of more than a hundred-fold excess
of tRNA and herring sperm DNA. This included the initial expression library
screen, which was based on a procedure that allowed successful isolation of
another RNA-binding protein, TRBP
(Gatignol et al., 1991).
Further, during the solution assays NaCl was present at the rather high
concentration of 350 mM. Second, the total number of basic residues (7 Lys and
1 Arg) in the K-rich region of the K4- mutants with drastically reduced RNA
binding is the same as in the strongly binding C-terminally truncated
hRUL1-666 variant; this was further corroborated by the absence of a signal on
the Nortwestern blots when a fragment from an unrelated protein containing the
sequence K2NK4EKSK (i.e. 8 Lys in 11 amino acids in
total) was run in parallel. Finally, the Northwestern signal from the
hRUL510-878 was at least partially resistant to a washing step with a solution
of 1 mg/ml of heparin (Konarska,
1989
; Pinol-Roma et al.,
1988
), and the extent of resistance was similar to that observed
for the core protein of HBV, which is an established nucleic-acid-binding
protein (Hatton et al., 1992
;
Nassal, 1992
). Hence the
central KKKTK element of the K-rich motif appears to be a critical part of a
larger RNA-binding domain.
Regarding binding specificity, our Northwestern assays using three
different RNA probes did not reveal a preference for HBV--containing
RNA, at least not under the conditions used. Although database searches
revealed stretches of multiple lysines in various predicted gene products,
including putative RNA helicases, no protein containing the identical motif,
let alone one with characterized RNA-binding capacity, was found that would
have allowed for inferences from the hRUL138-RNA interaction. Remotely,
however, the preponderance of Lys-residues resembles that of the arginines in
the arginine-rich RNA-binding motif present, for instance, in the HIV-1 Tat
protein. Peptides derived from this sequence bind specifically to TAR RNA,
mainly mediated by a single Arg-residue surrounded by other basic residues
that recognize a bulge; however, with about 10-fold lower affinity they also
interact with other RNAs (Calnan et al.,
1991
). Possibly, our assays measure a similar basal RNA-binding
activity of hRUL138. More quantitative binding studies using the same probe
RNAs might reveal more explicit differences. To this end, we have also tested,
using the in-solution binding assay, the ability of homopolymeric RNAs to
compete with binding of the labeled probe RNA as described in
Fig. 2C. Poly-A and poly-U at
10 µg/ml reduced binding by about 80% whereas ten-fold higher
concentrations of poly-G and poly-C were required for a similar reduction;
double-stranded poly-IC was ineffective even at this concentration (data not
shown). Hence there is some discrimination between different kinds of RNA,
which again argues against mere electrostatic binding; at present, however,
these data do not allow for clear-cut conclusions on the actual RNA targets of
hRUL138. Given its large size, the fragments used in the assays may only be
imperfect mimics of the entire protein; furthermore, other factors associating
with hRUL138 could affect its binding specificity in vivo. Altogether we
envisage two alternatives for physiological RNA binding by hRUL138: either it
has a truely broad selectivity or, more likely, there are specific RNA targets
that remain to be identified, possibly by similar approaches to those recently
used for finding target RNAs of the FMRP protein whose absence causes fragile
X syndrome (Brown et al., 2001
;
Darnell et al., 2001
).
Ubiquitin ligase activity of hRUL138
When complemented with ubiquitin (or methyl ubiquitin), E1, E2 and ATP,
both in vitro translated hRUL138 and bacterially expressed fragments
containing the RING-H2 domain were capable of self-ubiquitylation. Efficient
reaction required all of the aforementioned components and an intact RING
domain, as demonstrated by the inactivity of variants that lacked the RING
domain or carried a Cys to Ser mutation in the RING motif
(Bays et al., 2001). hRUL138 is
also capable of trans-ubiquitylation, as shown by the efficient ubiquitylation
of a RING-deleted variant upon co-expression in E. coli. Plausibly,
the spatial proximity between the E2-E3 complex and the target protein
required for trans-ubiquitylation was brought about by the ability of hRUL138
to undergo homomeric interactions, and these might be mediated by the
coiled-coil domain in the center of hRUL138. Whether homomeric interactions
between hRUL138 molecules exist in vivo and, if so, whether
trans-ubiquitylation from one to another molecule within the complex occurs
cannot be answered by these in vitro experiments. However, preliminary data
show that a non-related RNA-binding protein, selenocysteine-insertion-sequence
(SECIS)-RNA-binding protein 2 [SBP2
(Copeland et al., 2001
)] can
also be ubiquitylated by hRUL138. Together these data demonstrated that
hRUL138 meets the criteria of a genuine E3 enzyme. The physiological relevance
of the auto-ubiquitylation activity of hRUL138 is strongly supported by its
marked accumulation in transfected cells upon inhibition of
proteasome-dependent turnover, which, by contrast, had no effect on a
RING-deficient variant. Since the latter was expressed much better even
without proteasome inhibition, it is likely that the authentic protein is
subject to RING-mediated autoregulation as, for example, reported for the RING
E3 Mdm2 (Fang et al.,
2000
).
Expression and intracellular localization of hRUL138
Northern blotting revealed a weak hRUL138-specific band of about 4.8 kb in
all tissues tested, with the highest intensity in skeletal muscle followed by
heart, kidney and brain, and finally pancreas, lung, liver and placenta. These
data agree well with RT-PCR-derived semiquantifications for KIAA0675 RNA
(available at:
http://www.kazusa.or.jp/huge/).
In that study, the highest levels (10-50 fg/ng specific plasmid DNA
equivalents per ng poly-A+ RNA) were found in the heart, skeletal
muscle, kidney and testis, lower amounts (1-10 fg/ng poly-A+ RNA)
were found in the brain and very low levels (<1 fg/ng poly-A+
RNA) in the lung, liver, pancreas, spleen and ovary. Hence hRUL138 mRNAs are
expressed at very low levels; the same is probably true for the protein.
Whether the higher levels of hRUL138 mRNA in skeletal muscle and heart are
related to the low proliferative activity of these tissues is unclear;
notably, two other ubiquitin ligases, MuRF1 and MAFbx, are also predominantly
expressed in these tissues and have been shown to be critically involved in
muscle atrophy (Bodine et al.,
2001). However, hRUL138 mRNA expression may also be inducible in
these and in other tissues and, in addition, be regulated on the protein level
(see above). Two larger cross-reactive bands of about 5.5 kb and 7.5 kb were
detected in some tissues. Given that several of the cDNA clones obtained by
molecular hybridization contained intronic sequences, these larger mRNAs are
probably derived from alternative splicing events. In two of the clones, for
instance, a 805 bp intron is not spliced out between positions 1962 and 1963
of the hRUL138 ORF (see Fig.
1A); the larger mRNAs might correspond to these cDNAs. Although
the full range of potential splicing variants remains to be explored it is
highly likely from the appearance of more than one hRUL mRNA species that
alternative splicing does occur physiologically, implying the existence of
various isoforms.
Regarding the intracellular distribution of hRUL138, the weakly expressed
full-length GFP fusion protein was clearly located outside the nucleus in what
appeared to be a structured cytoplasmic compartment. The RING-deleted variant
hRUL1-1128 gave much stronger signals with a very similar distribution. On the
basis of the similar appearance of cells transfected with an ER-targeted RFP2
variant these structures might represent the ER, or a subcompartment thereof,
because the two proteins did not fully colocalize and appeared to segregate
with time. This localization was apparently independent of the RING-H2 domain,
which was not too surprising given that examples for both RING-dependent and
RING-independent localization of RING proteins are known
(Hu and Fearon, 1999;
Shin et al., 2001
). We note
that this conclusion rests on the similar appearance of the truncated hRUL-GFP
proteins and that of the very few detectably GFP-positive cells transfected
with full-length hRUL138-GFP. We cannot rule out the possibility that these
few cells might be defective in ubiquitylation or proteasome-mediated
turnover. Hence it remains formally possible that, in a normal cellular
environment, an active hRUL138 RING domain does affect the protein's
localization. However, even a GFP fusion with hRUL510-878 had a very similar
intracellular distribution; hence, neither of the three weakly predicted
transmembrane helices is essential for localization. This suggests that
compartment association occurs, different from that of the
transmembrane-domain-containing ubiquitin ligases Hrd1p
(Bays et al., 2001
) or Tul1
(Reggiori and Pelham, 2002
),
in an indirect fashion, possibly via interactions mediated by the predicted
coiled-coil region (amino acids 600 to 950), which is part of the localizing
variant 510 to 878.
What is the physiological role of hRUL138?
As shown above, hRUL138 is a non-abundant RNA-binding RING-H2
ubiquitin-protein ligase that localizes to an intracytoplasmic compartment,
possibly the ER. At present we can only speculate about its physiological
function. The absence of homologs in lower eukaryotes not only prevents
straightforward genetic experiments but also indicates a function restricted
to higher organisms; together with its low abundance this makes a specialized
function more likely than a role in general cellular metabolism. In this
light, a relationship with ER-associated degradation (ERAD), a mechanism
involved in cellular quality control and regulation of normal ER-resident
proteins (Bays et al., 2001;
Fang et al., 2001
), does not
seem likely. That hRUL138 cooperates, in vitro, productively with UbcH5 but
not UbcH7 indicates the specificity of the E2-E3 interaction. However, each E2
enzyme can interact with more than one E3 and, in this respect, UbcH5 appears
to be relatively promiscuous, not permitting direct conclusions on the
physiological function of hRUL138. Eventually the natural RNA and protein
ubiquitylation targets will have to be identified; both are probably major
endeavours, especially when considering that the 138 kDa protein is probably
only one out of several splicing variants. Perhaps the most intriguing and at
the same time tractable question is whether the RNA-binding and the
ubiquitylation activity of hRUL138 are functionally related. If so, hRUL138
may be an E3 enzyme that achieves target specificity using RNA as a mediator.
In the auto-ubiquitylation and homo-oligomer-based trans-ubiquitylation assays
described above we did not find an influence of RNA on ubiquitylation
activity; however, in preliminary studies hRUL138-mediated ubiquitylation of
SBP2 was markedly enhanced by the presence of RNA. Although the underlying
mechanism remains to be elucidated this suggests that RNA-binding proteins or
ribonucleoprotein particles (RNPs) might be physiological targets of hRUL138;
whether this includes RNPs derived from HBV, or other viruses, remains to be
determined.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bays, N. W., Gardner, R. G., Seelig, L. P., Joazeiro, C. A. and Hampton, R. Y. (2001). Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER- associated degradation. Nat. Cell Biol. 3,24 -29.[CrossRef][Medline]
Blumberg, B. S. (1997). Hepatitis B virus, the
vaccine, and the control of primary cancer of the liver. Proc.
Natl. Acad. Sci. USA 94,7121
-7125.
Bodine, S. C., Latres, E., Baumhueter, S., Lai, V. K., Nunez,
L., Clarke, B. A., Poueymirou, W. T., Panaro, F. J., Na, E., Dharmarajan, K.
et al. (2001). Identification of ubiquitin ligases required
for skeletal muscle atrophy. Science
294,1704
-1708.
Borden, K. L. (2000). RING domains: master builders of molecular scaffolds? J. Mol. Biol. 295,1103 -1112.[CrossRef][Medline]
Borden, K. L. and Freemont, P. S. (1996). The RING finger domain: a recent example of a sequence-structure family. Curr. Opin. Struct. Biol. 6, 395-401.[CrossRef][Medline]
Brown, V., Jin, P., Ceman, S., Darnell, J. C., O'Donnell, W. T., Tenenbaum, S. A., Jin, X., Feng, Y., Wilkinson, K. D., Keene, J. D. et al. (2001). Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107,477 -487.[Medline]
Calnan, B. J., Tidor, B., Biancalana, S., Hudson, D. and Frankel, A. D. (1991). Arginine-mediated RNA recognition: the arginine fork. Science 252,1167 -1171.[Medline]
Cheng, A. C., Calabro, V. and Frankel, A. D. (2001). Design of RNA-binding proteins and ligands. Curr. Opin. Struct. Biol. 11,478 -484.[CrossRef][Medline]
Copeland, P. R., Stepanik, V. A. and Driscoll, D. M.
(2001). Insight into mammalian selenocysteine insertion: domain
structure and ribosome binding properties of Sec insertion sequence binding
protein 2. Mol. Cell. Biol.
21,1491
-1498.
Daher, A., Longuet, M., Dorin, D., Bois, F., Segeral, E.,
Bannwarth, S., Battisti, P. L., Purcell, D. F., Benarous, R., Vaquero, C. et
al. (2001). Two dimerization domains in the trans-activation
response RNA-binding protein (TRBP) individually reverse the protein kinase R
inhibition of HIV-1 long terminal repeat expression. J. Biol.
Chem. 276,33899
-33905.
Darnell, J. C., Jensen, K. B., Jin, P., Brown, V., Warren, S. T. and Darnell, R. B. (2001). Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107,489 -499.[Medline]
Fang, S., Ferrone, M., Yang, C., Jensen, J. P., Tiwari, S. and
Weissman, A. M. (2001). The tumor autocrine motility factor
receptor, gp78, is a ubiquitin protein ligase implicated in degradation from
the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA
98,14422
-14427.
Fang, S., Jensen, J. P., Ludwig, R. L., Vousden, K. H. and
Weissman, A. M. (2000). Mdm2 is a RING finger-dependent
ubiquitin protein ligase for itself and p53. J. Biol.
Chem. 275,8945
-8951.
Freemont, P. S. (2000). RING for destruction? Curr. Biol. 10,R84 -R87.[CrossRef][Medline]
Gatignol, A., Buckler-White, A., Berkhout, B. and Jeang, K. T. (1991). Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science 251,1597 -1600.[Medline]
Hatton, T., Zhou, S. and Standring, D. N. (1992). RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J. Virol. 66,5232 -5241.[Abstract]
Hershko, A. and Heller, H. (1985). Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates. Biochem. Biophys. Res. Commun. 128,1079 -1086.[Medline]
Hicke, L. (2001). A new ticket for entry into budding vesicles - ubiquitin. Cell 106,527 -530.[Medline]
Hu, G. and Fearon, E. R. (1999). Siah-1
N-terminal RING domain is required for proteolysis function, and C-terminal
sequences regulate oligomerization and binding to target proteins.
Mol. Cell. Biol. 19,724
-732.
Ishikawa, K., Nagase, T., Suyama, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N. and Ohara, O. (1998). Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 5,169 -176.[Medline]
Kikuno, R., Nagase, T., Suyama, M., Waki, M., Hirosawa, M. and
Ohara, O. (2000). HUGE: a database for human large proteins
identified in the Kazusa cDNA sequencing project. Nucleic Acids
Res. 28,331
-332.
Konarska, M. M. (1989). Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol. 180,442 -453.[Medline]
König, S., Beterams, G. and Nassal, M.
(1998). Mapping of homologous interaction sites in the hepatitis
B virus core protein. J. Virol.
72,4997
-5005.
Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama,
S. and Weissman, A. M. (1999). RING fingers mediate
ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc.
Natl. Acad. Sci. USA 96,11364
-11369.
Lupas, A. (1996). Coiled coils: new structures and new functions. Trends Biochem. Sci. 21,375 -382.[CrossRef][Medline]
Mastrandrea, L. D., You, J., Niles, E. G. and Pickart, C. M.
(1999). E2/E3-mediated assembly of lysine 29-linked polyubiquitin
chains. J. Biol. Chem.
274,27299
-27306.
Nassal, M. (1992). The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive- strand DNA synthesis but not for virus assembly. J. Virol. 66,4107 -4116.[Abstract]
Nassal, M. (1999). Hepatitis B virus replication: novel roles for virus-host interactions. Intervirology 42,100 -116.[CrossRef][Medline]
Nassal, M. (2000). Macromolecular interactions in hepatitis B virus replication and particle assembly. In DNA virus replication, vol. 26 (ed. A. J. Cann), pp. 1-40. Oxford, UK: Oxford University Press.
Nuber, U., Schwarz, S., Kaiser, P., Schneider, R. and Scheffner,
M. (1996). Cloning of human ubiquitin-conjugating enzymes
UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP
and RSP5. J. Biol. Chem.
271,2795
-2800.
Nuber, U., Schwarz, S. E. and Scheffner, M. (1998). The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate. Eur. J. Biochem. 254,643 -649.[Abstract]
Ohira, M., Ootsuyama, A., Suzuki, E., Ichikawa, H., Seki, N., Nagase, T., Nomura, N. and Ohki, M. (1996). Identification of a novel human gene containing the tetratricopeptide repeat domain from the Down syndrome region of chromosome 21. DNA Res. 3, 9-16.[Medline]
Palombella, V. J., Rando, O. J., Goldberg, A. L. and Maniatis, T. (1994). The ubiquitin-proteasome pathway is required for processing the NF- kappa B1 precursor protein and the activation of NF-kappa B. Cell 78,773 -785.[Medline]
Perez-Canadillas, J. M. and Varani, G. (2001). Recent advances in RNA-protein recognition. Curr. Opin. Struct. Biol. 11,53 -58.[CrossRef][Medline]
Perri, S. and Ganem, D. (1996). A host factor that binds near the termini of hepatitis B virus pregenomic RNA. J. Virol. 70,6803 -6809.[Abstract]
Perri, S. and Ganem, D. (1997). Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis. J. Virol. 71,8448 -8455.[Abstract]
Pickart, C. M. (2000). Ubiquitin in chains. Trends Biochem. Sci. 25,544 -548.[CrossRef][Medline]
Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70,503 -533.[CrossRef][Medline]
Pickart, C. M. and VanDemark, A. P. (2000). Opening doors into the proteasome. Nat. Struct. Biol. 7,999 -1001.[CrossRef][Medline]
Pinol-Roma, S., Choi, Y. D., Matunis, M. J. and Dreyfuss, G. (1988). Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 2,215 -227.[Abstract]
Reggiori, F. and Pelham, H. R. (2002). A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies. Nat. Cell Biol. 4, 117-123.[CrossRef][Medline]
Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and Goldberg, A. L. (1994). Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78,761 -771.[Medline]
Saurin, A. J., Borden, K. L., Boddy, M. N. and Freemont, P. S. (1996). Does this have a familiar RING? Trends Biochem. Sci. 21,208 -214.[CrossRef][Medline]
Shin, M. E., Ogburn, K. D., Varban, O. A., Gilbert, P. M. and
Burd, C. G. (2001). FYVE domain targets Pib1p ubiquitin
ligase to endosome and vacuolar membranes. J. Biol.
Chem. 276,41388
-41393.
Small, I. D. and Peeters, N. (2000). The PPR motif a TPR-related motif prevalent in plant organellar proteins. Trends Biochem. Sci. 25,46 -47.[Medline]
Smith, C. A., Calabro, V. V. and Frankel, A. D. (2000). An RNA-binding chameleon. Mol. Cell 6,1067 -1076.[Medline]
Vogt, V. M. (2000). Ubiquitin in retrovirus
assembly: actor or bystander? Proc. Natl. Acad. Sci.
USA 97,12945
-12947.
Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J. and Chen, Z. J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412,346 -351.[CrossRef][Medline]
Weber, M., Bronsema, V., Bartos, H., Bosserhoff, A., Bartenschlager, R. and Schaller, H. (1994). Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription. J. Virol. 68,2994 -2999.[Abstract]
Zolotukhin, S., Potter, M., Hauswirth, W. W., Guy, J. and Muzyczka, N. (1996). A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70,4646 -4654.[Abstract]
Related articles in JCS:
|