Download A gene trap Dissociation insertion line, associated with a RING

Gene 290 (2002) 63–71
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A gene trap Dissociation insertion line, associated with a RING-H2 finger
gene, shows tissue specific and developmental regulated expression of the
gene in Arabidopsis
Esther Lechner a, Pierre Goloubinoff b, Pascal Genschik a,*, Wen-Hui Shen a,*
a
Institut de Biologie Moléculaire des Plantes du CNRS, 12, rue du Général Zimmer, 67084 Strasbourg Cédex, France
b
Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
Received 27 December 2001; accepted 11 March 2002
Received by B. Hohn
Abstract
Real interesting new gene (RING) finger proteins act as E3 ubiquitin-protein ligases and play critical roles in targeting the destruction of
proteins of diverse functions in all eukaryotes, ranging from yeast to mammals. Arabidopsis genome contains a large number of genes
encoding RING finger proteins. In this report we describe the identification of more than 40 RING-H2 finger proteins that are of small size,
not more than 200 amino acids, and contain no other recognizable protein–protein interaction domain(s). We characterize RHA2b, one of
these small RING-H2 finger genes. A gene trap line, SGT6304, was identified to contain a Dissociation (Ds) insertion in RHA2b gene. No
RHA2b transcript was detected in the homozygous SGT6304 plants. Despite the elimination of RHA2b function, homozygous SGT6304
plants lacked detectable growth or development defects, suggesting functional redundancy of RHA2b with other RING finger genes.
Expression of RHA2b was specifically active in vascular tissue and in upper pistil of inflorescence as well as in root tip and shoot apical
meristem region. Potential functions of ubiquitin-proteolysis pathway in vascular formation and in fertilization are discussed. q 2002
Elsevier Science B.V. All rights reserved.
Keywords: Proteolysis; Ubiquitin ligase; Vascular formation; Transposon tagging
1. Introduction
The RING (for Real Interesting New Gene) finger motif is
a small zinc-binding domain defined by the consensus
sequence Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)Cys/His-X2-Cys-X(4–48)-Cys-X2-Cys, where X can be
any amino acids and the Cys (cysteine) and His (histidine)
representing zinc binding residues. RING fingers can be
subcategorized into RING-HC and RING-H2 depending
of whether a Cys or His occupies the fifth coordination
site, respectively. The RING finger forms one integrated
structural unit which binds two zinc atoms in a unique
Abbreviations: RING, real interesting new gene; Cys (C), cysteine; His
(H), histidine; Ub, ubiquitin; Km, kanamycin; SCF, Skpl Cullin F-box
protein complex; APC, anaphase-promoting complex; Ds, dissociation;
PCR, polymerase chain reaction; GUS, b-glucuronidase; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal; BY2, bright yellow 2; HSP, heat
shock protein; CDK, cyclin-dependent kinase
* Corresponding authors. Tel.: 133-3-8841-7200; fax: 133-3-88614442.
E-mail addresses: [email protected]
(P. Genschik), [email protected] (W.-H. Shen).
‘cross-brace’ arrangement, differing from the tandem
arrangement of zinc binding sites characteristic of zinc
fingers (reviewed in Freemont, 2000; Joazeiro and Weissman, 2000).
Recent studies demonstrate that RING finger proteins
play critical roles in specific ubiquitination events and it
has been more generally hypothesized that all RING
proteins act as ubiquitin (Ub) protein ligases (Freemont,
2000; Joazeiro and Weissman, 2000). Ub-dependent proteolysis pathway controls the selective degradation of a large
number of proteins involved in diverse cellular processes
that include cell cycle regulation, stress responses, signal
transduction, metabolic regulation and cell differentiation
(for review, see Hershko and Ciechanover, 1998). Generally, protein ubiquitination is achieved through sequential
action of three types of enzymes: Ub-activating enzyme
(E1), Ub-conjugating enzyme (E2) and Ub-protein ligases
(E3). Ub is first attached, through its C-terminus, to the
active site cysteine of E1; then transferred to an E2, again
through a thiol-ester linkage. E3s, which are primarily
responsible for providing specificity to Ub conjugation,
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.
PII: S 0378-111 9(02)00556-5
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E. Lechner et al. / Gene 290 (2002) 63–71
interact with E2 and substrate, facilitating formation of
isopeptide linkages between the C-terminus of Ub and
lysines either on a target protein or on the last Ub of a
substrate-bound poly-Ub chain. Once a poly-Ub chain is
assembled on a substrate, the substrate is then degraded
by the 26S proteasome. The 26S proteasome appears to be
similar in organization and structure, and likely functions in
an analogous manner in plant as in animal cells (Parmentier
et al., 1997).
RING finger motifs have been shown in many cases to be
required for interactions between RING finger proteins and
E2s (reviewed in Freemont, 2000; Joazeiro and Weissman,
2000). Crystal structure analysis revealed that the E2
UbcH7 binds to the RING protein c-Cbl through contacts
between a groove within the RING domain of c-Cbl and two
loops in the E2 fold of UbcH7 (Zheng et al., 2000).
Although elements determining the specificity of RINGE2 pairs have yet to be clearly defined, RING fingers have
been proposed to serve to specifically recruit and allosterically activate E2s.
The recruitment of substrates by the RING finger E3s can
be achieved either through additional protein–protein interaction domains present on the RING proteins themselves,
such as the SH2-domain in the case of c-Cbl (Joazeiro and
Weissman, 2000), or through multisubunit protein
complexes. A small noncanonical RING-H2 finger protein,
RBX1 (also referred to as ROC1 and HRT1), has been
identified to be an essential component of SCF E3
complexes (reviewed in Tyers and Jorgensen, 2000). The
SCF complex, named from the three initially identified
subunits SKP1, Cullin/CDC53, F-box protein, targets a
number of cell cycle regulators, transcription factors and
other proteins for degradation. In this complex, the F-box
protein acts as substrate-targeting subunit and interacts
through the SKP1 intermediate with the N-terminal part of
the Cullin/CDC53. RBX1 promotes association of the E2
CDC34 with the complex through interaction with the Cterminus of the Cullin/CDC53 (Wu et al., 2000; Furukawa
et al., 2000). In a strikingly similar manner, the small RINGH2 finger protein APC11 is an essential component of the
APC (or cyclosome) E3 complex, which specifically targets
a number of mitotic factors such as cyclin B and securin for
degradation (Gmachl et al., 2000; Leverson et al., 2000).
Sequence similarity analysis reveals that the Arabidopsis
genome, the first entirely sequenced plant genome (The
Arabidopsis Genome Initiative, Nature 408: 796–815),
contains a large number of genes encoding RING finger
proteins. Although characterization of several plant RING
finger proteins including the photomorphogenesis regulators
COP1 and CIP8 (Torii et al., 1999), the N-end rule E3
enzyme PRT1 (Potuschak et al., 1998), and the
membrane-bound E3 enzyme RMA1 (Matsuda et al.,
2001) have been reported, few molecular data are available
on the expression and function of the small RING finger
proteins. In this report, we describe characterization of the
Arabidopsis RHA2b gene (named according to Jensen et al.,
1998) that encodes a RING-H2 finger protein of 147 amino
acids. A Dissociation (Ds) insertion line associated with
RHA2b was initially isolated during random analysis of
flanking sequences from the collection of gene trap lines
(Parinov et al., 1999). Analysis of this Ds-insertion line
revealed that the Ds-insertion has created a loss-of-function
allele. No obvious mutant phenotype was evident at the
studied experimental conditions, suggesting redundant
function of RHA2b with other genes. Reporter gene expression patterns revealed that RHA2b was highly expressed in
vascular tissues as well as in the transmitting tissue of the
styles of flowers.
2. Materials and methods
2.1. Plant materials
The Arabidopsis plants were of the landsberg erecta
ecotype. Seeds were produced under greenhouse conditions.
Arabidopsis cell suspensions were maintained by weekly
subculture as described by Glab et al. (1994).
2.2. Arabidopsis line SGT6304
Arabidopsis line SGT6304 containing Ds insertion in the
RHA2b gene was kindly provided by Dr. V. Sundaresan
(Institute of Molecular Agrobiology, The National University of Singapore). F4 seeds collected from multiple kanamycin (Km) resistant plants were surface sterilized and plated
onto Km (50 mg/l)-supplemented medium (half-strength
Murashige and Skoog salts, 1% sucrose, 0,9% agar, pH
5.7). Km-resistant plants were transplanted to soil and
allowed to set seeds by self-fertilization. Homozygous
plants, which segregated 100% for Km resistance, were identified in the F4. Ds integration in the RHA2b gene was
confirmed by PCR-amplification using gene specific primers:
5 0 -AGCTTAGATAGAGATATGAAATCAGCAT-3 0
(corresponding to 2293 to 2265 bp upstream of ATG codon
of RHA2b) and 5 0 -TCGTTTCCGTCCCGCAAGT-3 0 (Ds3 0 3a in Parinov et al., 1999) for the 5 0 -end, and 5 0 -TTGAGTTTAGACAGACACGATGCAGT-3 0 (corresponding to 1295
to 1323 bp downstream of ATG codon of RHA2b) and 5 0 TCCGTTCCGTTTTCGTTTTTTAC-3 0 (Ds5 0 -2a in Parinov
et al., 1999) for the 3 0 -end junction between RHA2b and the
Ds. The PCR products were sequenced to determine the
precise fusion between RHA2b and Ds.
2.3. Histochemical GUS assays
Whole-mount b-glucuronidase (GUS)-staining of Arabidopsis seedlings germinated on plates or inflorescences
from soil-grown plants were performed using the method
described by Jefferson et al. (1987). For visualization, the
tissues were cleared for chlorophyll by rinsing with ethanol
(70–95%) and photographed under a stereomicroscope or
with Nomarski optics. Thin sections of GUS-stained mate-
E. Lechner et al. / Gene 290 (2002) 63–71
rial were prepared after embedding in Historesin according
to the manufacturer’s recommendations (Leica Instruments
GmbH, Heidelberg). Images were scanned and processed
with Adobe Photoshop 3.0 (Adobe systems, Mountain
View, CA).
2.4. Treatments of plant tissues and suspension cultured
cells
RHA2b expression in response to the plant hormone auxin
was assayed according to the method described by Gil et al.
(1994). Briefly, hypocotyls of etiolated seedlings of Arabidopsis that had been grown in complete darkness for 10 days
were cut into 2–3-mm sections. After incubation in KPSC
buffer for 4 h to deplete endogenous hormones, the
segments were transferred to fresh KPSC buffer with or
without exogenous hormones for 1 h at 28 8C and then
frozen in liquid nitrogen for RNA preparation.
The leaf-strips experiments used to study RHA2b expression during re-initiation of cell division activity in Arabidopsis have been previously described (Genschik et al., 1994).
Carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) has
been previously shown to be an efficient proteasome inhibitor that induces metaphase arrest in tobacco BY2 cells
(Genschik et al., 1998). To test RHA2b expression in
response to MG132, 3-day-old suspension cultured cells
of Arabidopsis were incubated with 100mM MG132. Cells
were collected 1 and 3 h after the treatment and frozen in
liquid nitrogen for RNA preparation.
2.5. Northern blot analysis
Total RNA was isolated from various plant tissues as well
as from suspension cultured cells via the hot phenol method
(Nagy et al., 1988). Aliquots of 20 mg RNA were analyzed
by electrophoresis on formaldehyde/agarose gels and
blotted onto Hybond-NX nylon membranes according to
the manufacturer’s recommendations (Amersham Pharmacia Biotech). Hybridization was performed under standard
high-stringency conditions (Sambrook et al., 1989). The
RHA2b and HSP70 probes correspond to the EST clones
11A10TT (GenBank AA728515) and G5C8T7 (GenBank
N96136), respectively. The SAUR probe corresponds to
the PCR-amplified genomic fragment of the SAUR-AC1
gene (Gil et al., 1994). The UBI probe was prepared from
the clone pHUb14-38 containing the human ubiquitin
cDNA (Genschik et al., 1994). The integrity and the amount
of the loaded RNA were verified by staining with ethidium
bromide or hybridization with an Arabidopsis 25S rRNA
probe (GenBank T44938).
2.6. Yeast two-hybrid analysis
The entire coding region of RHA2b was PCR-amplified
using the 5 0 -end primer 5 0 -TCAGAATTCATGAGATGCAAGGTAGTATATGCAGCAAGAACAACAACAAGAAC-3 0 and the 3 0 -end primer 5 0 -ACAGGATCCTCAA-
65
TGAGATGATGCAGTAGAGGTA-3 0 , and subsequently
cloned into the EcoRI and BamHI sites of pGBKT7 vector
(Clontech Laboratories Inc., Palo Alto, CA). The derived
plasmid was sequenced to confirm the absence of errors
from PCR-amplification and the correct in-frame fusion
with the GAL4 DNA binding domain. Plasmids containing
Arabidopsis Cullin cDNAs fused with the GAL4 activation
domain were obtained (W.-H. Shen, unpublished). Interactions between RHA2b and Cullins were assayed according
to the manufacturer’s recommendations (Clontech).
2.7. Sequence analysis
Database searches were performed using BLASTP
(http://www.ncbi.nlm.nih.gov/BLAST/). Multiple alignments of protein sequences were done with the ClustalW
program (http://www.ebi.ac.uk/clustalw/) and phylogenetic
tree displayed using TreeViewPPC (Roderic D.M. Pase)
programs.
3. Results
3.1. Homozygous SGT6304 line plants fail to express
RHA2b RNA,but appear phenotypically normal
An insertional mutagenesis system through use of Ds/Ac
transposable elements had been developed in Arabidopsis by
Sundaresan et al. (Parinov et al., 1999). In this system, the
modified Ds element carrying the GUS reporter gene as well
as the NPTII gene that confers resistance to Km as a selectable marker was used as a gene trap. Random analysis of
sequences flanking the Ds had revealed that the SGT6304
line contains Ds insertion associated with the RHA2b gene
(Parinov et al., 1999). From F4 progeny, we selected both
heterozygous and homozygous SGT6304 lines, which exhibited the characteristic segregation ratios for Km-resistance.
PCR-amplification with Ds and RHA2b specific primers
confirmed Ds insertion in the RHA2b gene. Sequence analysis of the PCR-amplified products revealed that the Ds is
inserted at the very N-terminal coding region, between the
fourth and fifth codon, of the RHA2b gene (Fig. 1A).
Northern blot analysis detected a single band with the
predicted size. This transcript could be detected only in
heterozygous but not in homozygous SGT6304 plants
(Fig. 1B), indicating that the Ds insertion created a lossof-function allele of RHA2b. Despite the null mutation of
RHA2b, homozygous SGT6304 plants do not exhibit
obvious growth or developmental defects. The mutant
seeds germinate at the same time as wild-type ones, and
the plants show normal morphology. Flowering time and
seed production are also not affected.
3.2. RHA2b transcript accumulates at different levels in
several types of plant tissues
To determine the tissue distribution of RHA2b transcript,
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E. Lechner et al. / Gene 290 (2002) 63–71
Fig. 1. (A) Schematic representation of the Ds insertion in the RHA2b gene
in SGT6304 line. The promoterless GUS gene begins 343 nt from the end of
Ds. Inside of the Ds localizes also the NPTII gene under the control of the
CaMV 35S promoter that confers resistance to kanamycin. Arrow-lines
represent the different promoter regions and open boxes the coding
sequence of different proteins. (B) Northern blot analysis of RHA2b expression in SGT6304 plants. Total RNA were prepared from 2-week-old heterozygous (lane 1) and homozygous (lane 2) plants. Top panel shows
hybridization with RHA2b probe and lower panel shows ethidium
bromide-stained gel as loading control.
we performed northern blot analysis with total RNA from
siliques, flowers, leaves, stems and roots (Fig. 2). RHA2b is
expressed at higher levels in flowers and roots, and at lower
levels in stems, leaves and siliques. Only a very weak hybridization signal could be detected in suspension cultured
cells (Fig. 2). The heat shock HSP70 gene exhibited a different pattern of expression, which is with high levels in
suspension cultured cells, in siliques and flowers whereas
low in roots (Fig. 2).
3.3. Reporter gene expression patterns in SGT6304 line
plants reveal tissue specificity of RHA2b expression
The gene trap Ds carries the promoterless GUS gene whose
expression relies entirely on transcription signals of the
tagged gene. In the SGT6304 line, the GUS gene was fused
in the transcribed orientation with the RHA2b gene (Fig. 1A).
Thus, the GUS gene represents an ideal reporter for studying
the expression pattern of the tagged RHA2b gene.
Both in vitro- and greenhouse-grown plants were
analyzed for GUS activity by histochemical staining. No
significant difference in GUS staining pattern was noticed
between in vitro-grown and greenhouse-grown plants. At
juvenile phase, GUS staining was consistently localized in
the vascular cylinder throughout the entire seedling (Fig.
3A). The strongest GUS staining was found in meristematic
regions of shoots (Fig. 3B), in hypocotyl-root transition
zones (Fig. 3C), and in tips of primary and lateral roots
(Fig. 3D,F). A relatively strong staining was also observed
in the vascular cylinder regions where lateral roots emerged
(Fig. 3E,F).
At the adult phase, vasculature-localized GUS staining
could still be observed but preferentially in young organs,
such as upper axillary shoots and stems close to inflorescences (data not shown). In inflorescences, the strongest
GUS staining was detected in the upper pistil, from floral
stage 11 (Smyth et al., 1990) onward (Fig. 3G). Particular
strong staining was localized to the lower stigmatic zone and
the upper transmitting tract, which is composed of the longitudinal files of cells, of the pistil (Fig. 3H,I). The outer
papillae of the stigma was barely stained. With prolonged
incubation time, GUS staining was also visible on sepals of
flowers (data not shown). Upon fertilization the strong GUS
staining extended from the upper pistil to the carpel walls,
and localized mainly in the valve margin and replum of the
walls (Fig. 3J). The staining of carpel walls disappeared after
the developmental time corresponding to heart embryo stage.
In ovaries, GUS staining was detected in embryo-developing
areas at early stages, up to early heart stage (Fig. 3K). GUS
staining in embryos was observed at mature stage (Fig. 3L),
with strongest staining in the tip of radicle (Fig. 3M).
Together these GUS staining patterns reveal that the
expression of RHA2b is particularly active in shoot and
root tips, in vascular tissues of various organs, and in
upper pistils of inflorescence. This is consistent with the
previous Northern data and further suggests that RHA2b
functions in a highly tissue-specific and developmental
regulated manner.
3.4. Accumulation of RHA2b transcript in response to
auxins and MG132
Fig. 2. Northern blot analysis of RHA2b expression in different organs of
plants and in suspension cultured cells. Total RNA were isolated from
different organs of Arabidopsis plants as well as from suspension cultured
cells at different days of subculture, as indicated. Northern analysis was
performed by successive hybridizations with RHA2b and HSP70 probes.
Ethidium bromide-stained image is shown as loading control.
It is well established that the phytohormone auxin play
essential role in lateral root primordium initiation (Celenza
et al., 1995; and references therein). The strong GUS staining detected in root tips and around lateral root initiation
sites in SGT6304 plants promoted us to evaluate the regulation of RHA2b expression by auxin. We chose the experimental system of using hypocotyls of etiolated seedling
because of its rapid response to auxin (Gil et al., 1994).
E. Lechner et al. / Gene 290 (2002) 63–71
67
Fig. 3. Histochemical localization of GUS activity in the gene trap line SGT6304. (A) One-week-old seedling. (B) Shoot apical meristem region. (C)
Hypocotyl-root transition zone. (D) Primary root tip. (E) Lateral root initiation site (indicated by arrow). (F) Lateral root. (G) Inflorescences and early
developing siliques. (H) Longitudinal section of a GUS-stained upper pistil. (I) Close-up image of the upper transmitting tract of the pistil. (J) Close-up image
on the carpel wall of a young silique. (K) Embryo sac. (L) Mature embryo. (M) Close-up image on the tip of embryonic root. The same pattern was observed in
more than ten independent individuals.
The level of RHA2b transcript remained unchanged 1 h after
the treatment with auxins whereas the positive control
SAUR gene responded positively to the treatments (Fig.
4A). It is thus unlikely that RHA2b belongs to auxin-responsive gene family.
RHA2b expression during re-initiation of mitotic activity
was evaluated by using the leaf-strips experimental system
(Genschik et al., 1994). In this system the differentiated
leaf cells were stimulated to re-enter the cell cycle by
wounding and auxin stimulation. The level of RHA2b transcript decreased significantly at 24 h, and then recovered to
the initial value 48 and 72 h after the leaf strips culture
(Fig. 4B). The cell cycle gene histone H4 mRNAs,
however, accumulated to the highest level 24–48 h after
the culture (Fig. 4B), which parallels DNA synthesis
detected by [ 3H]dTTP incorporation (data not shown).
Thus RHA2b behaved distinctly from the histone H4
gene in exhibiting a transient down-regulation upon reenter the cell cycle.
To evaluate RHA2b expression in response to proteolysis inhibition, we performed Northern analysis on Arabi-
dopsis suspension cultured cells that had been treated with
MG132, an efficient and highly specific proteasome inhibitor (Genschik et al., 1998). An increased accumulation
of RHA2b transcript was observed 1 h after MG132 treatment (Fig. 4C). This effect seems transient since RHA2b
transcript level returned to the initial value 3 h after the
treatment. Polyubiquitin transcripts also accumulated, but
with a different pattern that increased with prolonged
MG132 treatment. In contrast, the level of the heat
shock HSP70 transcript remained unchanged upon
MG132 treatment.
3.5. Arabidopsis genome contains a large number of genes
encoding small RING-H2 finger proteins
We searched in the database for sequence similar to the
RING-H2 domain of RHA2b. At least 101 Arabidopsis
proteins show significant matches with the RING-H2
domain. Among them 40 proteins are with a size of no
longer than 200 amino acids, a characteristic previously
used to define RHA group (Jensen et al., 1998). Four
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E. Lechner et al. / Gene 290 (2002) 63–71
Fig. 4. Northern blot analysis of RHA2b expression in response to external stimuli. (A) Effects of auxins. Arabidopsis hypocotyl sections were incubated in the
absence (control) or in the presence of 11 mg/l 2,4-D or 1.8 mg/l IAA for 1 h, and then used for total RNA preparation. Hybridizations were performed with the
indicated probes. (B) Response upon re-initiation of mitotic activity from leaf-strips. Arabidopsis leaf-strips at different time points of subculture, as indicated,
were analyzed. Hybridizations with RHA2b and H4 probes as well as ethidium bromide-stained images were shown. (C) Effects of MG132. Untreated (control)
and MG132-treated Arabidopsis suspension cells, as indicated, were analyzed for RHA2b, UBI and HSP70 expression. Ethidium bromide-stained image is
shown as loading control.
more Arabidopsis proteins were also identified to exhibit
significant sequence similarities with human and yeast
RBX1/APC11 proteins. Alignment of the entire sequence
of these small RING-H2 proteins revealed that some of
them form distinct phylogenetic groups, but a large number
of them are only distantly related (Fig. 5A). This sequence
diversity suggests that these small RING-H2 proteins
display a large spectrum of function in Arabidopsis.
The RING-H2 domain of RHA2b showed highest homologies with the RING-H2 domains of RHA2a and the
mammalian proteins Arkadia and RLIM (Fig. 5B). This
group of RING-H2 domains differs from that of RBX1
and to a less extend of APC11 by a remarkable shortened
distance between Cys2 and Cys3, which forms the loop
between the two zinc binding sites. They resemble closer
to the canonical RING fingers of several structure-determined proteins (Brzovic et al., 2001, and references
therein), indicating that they may associate with E2
enzyme(s) through a similar structure interaction.
In the region outside of the RING-H2 domain, RHA2a
was the only protein from the current databases that shows
significant homologies with RHA2b (Fig. 5C). Although
RBX1 and APC11 proteins also do not contain recognizable
protein–protein interaction domain(s) beside the RING-H2
finger, their interaction with Cullin/CDC53 proteins had
been demonstrated in the yeast two-hybrid system. To test
potential interaction of RHA2b with Cullin, we performed
the yeast two-hybrid assays. No significant interaction
between RHA2b and AtCUL1 could be detected (data not
shown). It is very unlikely that RHA2b functions in SCF E3
ligases.
4. Discussion
In this study we report the characterization of a small
RING-H2 finger gene from Arabidopsis. By sequence
comparison, the RING-H2 finger of RHA2b shows good
conservation to the canonical RING fingers, suggesting
that RHA2b may associate with an E2 enzyme through a
similar structure interaction.
Despite the fact that RING finger proteins play diverse
cellular functions, including gene expression, signal transduction, peroxisome biogenesis, DNA repair and recombination, and membrane vesicle sorting (Freemont, 2000;
Joazeiro and Weissman, 2000), elimination of RHA2b function does not result in an obvious mutant phenotype in
Arabidopsis plants. In gene trap line SGT6304 the Ds is
inserted in the N-terminal region of the RHA2b coding
sequence, and RHA2b transcript could not be detected in
the homozygous SGT6304 plants, suggesting that the Ds
insertion has disrupted transcription of the RHA2b gene.
Homozygous SGT6304 plants, however, do not exhibit
any growth or development defects. This failure to observe
a mutant phenotype is likely due to the redundancy of
RHA2b with other RING finger genes. From a large number
of RING-H2 finger genes identified in Arabidopsis, the gene
most similar to RHA2b is RHA2a. Using RT–PCR amplification, Jensen et al. (1998) had shown that RHA2a transcript
is present in seedlings, stems, flowers, leaves, roots and
siliques. However, it is presently unknown if RHA2a
expression overlaps RHA2b expression in a tissue- or celltype specific fashion.
A high level of RHA2b expression was found in vascular
E. Lechner et al. / Gene 290 (2002) 63–71
69
Fig. 5. Sequence analysis of Arabidopsis small RING-H2 finger proteins. (A) A phylogenetic tree of Arabidopsis proteins together with RBX1 and APC11 of
Saccharomyces cerevisiae, and Homo sapiens. (B) Sequence alignment of the RING-H2 fingers. (C) Sequence comparison between RHA2b and RHA2a. The
Cys/His residues forming the two zinc-binding sites are indicated by bold letters. Consensus symbols on top of the alignments indicate: ‘*’ for the identical or
conserved residues in all aligned sequences, ‘:’ and ‘.’ for the conserved and semi-conserved substitutions, respectively. Accession numbers in the database:
At4g11370 for RHA1a, At4g11360 for RHA1b, At1g15100 for RHA2a, AF078823 for RHA2b, NP_010276 for ScAPC11, NP_014508 for ScRBX1,
NP_057560 for HsAPC11, NP_055063 for HsRBX1. AAD34209 for MmRLIM, NP_291082 for MmArkadia.
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E. Lechner et al. / Gene 290 (2002) 63–71
tissue of various organs, especially of developing- or youngorgans including root tips and hypocotyls. The E2 enzyme
AtUBC1-3 gene as well as some Ub genes have been
previously reported to be highly expressed in vascular tissue
(Plesse et al., 2001 and references therein). However,
RHA2b is the first potential E3 enzyme showing specific
expression in vascular tissue. Immunohistochemical localization studies reveal that Ub/Ub-conjugates and the proteasome are present at high levels in vascular tissue
(Ingvardsen et al., 2001). Together these observations
imply an important role of Ub-dependent pathway in vascular bundle formation. In supporting this assumption, overexpression of a Ub variant, with Lys48 replaced by Arg, had
been reported to induce marked abnormalities in vascular
tissue (Bachmair et al., 1990). Moreover, Woffenden et al.
(1998) had shown that tracheary element differentiation
from Zinnia mesophyll cell cultures requires proteasome
activity.
It has been observed that pericycle cells of the vascular
cylinder divide and lateral root primordia form without
intervening mitotic quiescence (Dubrovsky et al., 2000).
Consistently, a subset of cell cycle genes, such as A-type
CDKs, A- and D-type cyclins, have been described to highly
express in vascular tissue (Burssens et al., 2000; Swaminathan et al., 2000 and references therein). It is generally
viewed that cyclin proteins are degraded through Ub-dependent proteolysis pathway, which represents a key point of
control of cell division (Koepp et al., 1999). Plant cyclins
appear also submitted to similar type of control (Genschik et
al., 1998; Criqui et al., 2000). The findings of components of
proteasome pathway in the same type of tissue thus fulfill
the requirement of cell division control. In addition to developmental control, lateral root formation can be initiated
from pericycle cells by environmental cues. Auxin is essential for lateral root primordium initiation (Celenza et al.,
1995). It is well documented that the SCF E3 pathway is
involved in auxin signaling (del Pozo and Estelle, 1999).
Although strong GUS staining was found in root tips and the
region of lateral root initiation, RHA2b expression was not
induced by auxin. It is likely that RHA2b plays rather a role
in later steps, such as vasculature initiation from meristematic cells.
RHA2b expressed very actively in upper pistils, which
localized to the lower stigmatic zone and the upper transmitting tract. This observation suggests that RHA2b may
play a role in fertilization. Function of Ub-dependent
proteolysis in fertilization is poorly documented. Nevertheless, it has been reported that Ub-dependent mechanisms are
involved in the recognition and elimination of defective
spermatozoa in mammals (Sutovsky et al., 2001). In plants,
inhibition of proteasome activity has been shown to inhibit
pollen growth in vitro (Speranza et al., 2001). Moreover, the
D-type cyclin CYCD3;2, representing potential substrate of
Ub-dependent proteolysis, has also been found to express,
similar to RHA2b, in the carpel walls, in the stigma and in
the transmitting tract of the pistils (Swaminathan et al.,
2000). Recently, we demonstrated that loss-of-function of
the cullin AtCUL1 gene reduced inheritance in both male
and female gametophytes (W.-H. Shen et al., submitted).
Interestingly, both male and female gametogenesis of the
mutant are morphologically normal, suggesting that
AtCUL1-based E3 ligases may involved in fertilization
process.
Our two-hybrid data revealed that RHA2b did not interact
with AtCUL1, suggesting that RHA2b functions independently from the SCF E3 ligases. Consistently, the RING-H2
domains were only distantly related between RHA2b and
RBX1 proteins. Besides the RING finger motif, no other
conserved protein–protein interaction domain(s) could be
identified from the sequences of RHA2b. Partners of
RHA2b, therefore, remain to be identified.
It has been reported in mammalian as well as in yeast
cells that proteasome inhibition leads to induction of heat
shock proteins through likely stabilization of transcriptional
regulators (Zhou et al., 1996). In contrast, such induction
was not observed for the Arabidopsis HSP70 gene.
However, in agreement with our previous observation in
BY2 cells (Genschik et al., 1998), MG132 treatment
induced Ub transcript accumulation in Arabidopsis suspension cultured cells. RHA2b responded to MG132 treatment
by also an increased mRNA accumulation, but with a transient pattern that distinct from the continuous accumulation
of Ub mRNA. Although functional significance of this
induction as well as mechanism by which this induction
occurred is unknown, RHA2b provides, in addition to Ub,
an other gene of the Ub-dependent proteolysis pathway that
respond to proteasome inhibition.
Acknowledgements
We are grateful to V. Sundaresan for providing the
SGT6304 line. We thank S. Grava for yeast two-hybrid
experiments, A. Capron and M.-C. Criqui for help in Northern analysis, P. Hammann for DNA sequencing, and M.
Bergdoll for help in sequence similarity analysis. E.L. is
supported by the Association Franco-Israélienne pour la
Recherche Scientifique et Technologique (AFIRST).
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