To characterize the function of UBE2E3, we developed and affinity purified a rabbit polyclonal antibody, α
-2E3, directed against the unique amino terminal, 58-amino-acid region of the enzyme. α
-2E3 specifically recognized recombinant GST-UBE2E3 by Western blot but not GST fusions of two closely related enzymes, UBE2E2 and UBE2E1 (). These three enzymes represent the human class III E2s. Their catalytic core domains are 95% identical, and they are distinguished from each other by their unique amino terminal extensions.18
Western blot of RPE-1 cell lysates with α
-2E3 detected a primary band of 23 kDa, the predicted size of the enzyme (). A comparable band was also detected in isolated retinas from C57BL6 mice (). The mouse counterpart of UBE2E3 is called UbcM2, and the proteins are 100% identical. Immunolabeling of asynchronously growing RPE-1 cells with α
-2E3 revealed that the enzyme localizes to the nucleus of interphase cells and is dispersed throughout mitotic cells ().
Figure 1 α-2E3 specifically recognizes UBE2E3, and the enzyme localizes to interphase nuclei in telomerase-immortalized retinal pigment epithelial cells (RPE-1s). (A) α-GST (left) and α-2E3 (right) Western blot of GST and GST-E2 fusion (more ...)
To directly address whether UBE2E3 is essential in RPE cell proliferation, we used a loss-of-function approach with siRNA to knockdown expression of the enzyme. RT-PCR and complementary Western blotting demonstrated that individual siRNA duplexes, as well as pools of duplexes, efficiently knocked down UBE2E3 mRNA () and suppressed expression of the enzyme (). The specificity of the knockdown was further established by showing that the siRNAs did not reduce expression of the closely related enzyme, UBE2E1 (data not shown).
Figure 2 Knockdown of UBE2E3 by siRNA causes a block of cell proliferation. (A) RNA was isolated from cells treated with duplexes (Du) 1 to 4 or with a mixture of the duplexes (Du 1+2+3+4). RT-PCR products were generated with UBE2E3- and importin-11-specific primers. (more ...)
Next, we assessed the consequences of UBE2E3 depletion on cell proliferation. RPE-1 cells were transfected with siRNA, collected by trypsinization at 1, 2, and 3 days after siRNA treatment and counted. Depletion of UBE2E3 dramatically reduced the number of cells () compared with cells treated with siCON, the control siRNA. The reduced proliferation was observed with pools of siRNA, as well as with individual siRNAs (data not shown). Propidium iodide staining and FACS analysis showed that the UBE2E3 knockdown cells had a similar cell cycle distribution profile as control cells (data not shown) indicating that depletion of the enzyme was probably inducing a cell cycle exit. To confirm this, we immunolabeled control and knockdown cells for Ki-67, a marker of proliferating cells.25
We predicted that if the knockdown cells were arrested in the cell cycle, their nucleoli would stain positive for Ki-67, whereas if they had exited the cell cycle, they would be negative for the proliferation marker.26
Control siRNA-treated cells were proliferating as indicated by Ki-67 immunolabeling (). Depletion of the enzyme by either of two different siRNA duplexes led to a loss of Ki-67-positive cells (). These data show that UBE2E3 depletion causes cells to exit the cell cycle. Consistent with this exit, we also observed that knockdown of UBE2E3 with either duplex 2 or 3 caused an increase in cell spreading and flattening (). Cell area measurements showed that depletion of UBE2E3 caused an average twofold increase in area compared with siCON-treated cells ().
Figure 3 UBE2E3 knockdown causes RPE-1 cells to exit from the cell cycle and flatten. (A) Immunofluorescence analysis of RPE-1 cells treated with either siCON or UBE2E3-specific siRNA (duplexes 2 and 3). Two days after siRNA treatment, the cells were colabeled (more ...)
To further elucidate the mechanism(s) by which UBE2E3 depletion leads to an exit from the cell cycle, we analyzed knockdown cells for levels of the cell cycle inhibitor p27Kip1
is degraded by the ubiquitin proteolytic system during G1
, and this degradation is required for progression from the G1
to the S phase,7
as well as from the G2
to M phase.6
In addition, p27Kip1
accumulation can drive cells out of the cell cycle, and elevated levels of the protein are necessary to maintain a postmitotic state.28
In proliferating RPE-1 cells, p27Kip1
expression is maintained at relatively low levels (e.g., ). In contrast, cells depleted of UBE2E3 exhibited a robust increase in the percentage of p27Kip1
-positive cells as well as in the level of p27Kip1
per cell. This elevation of p27Kip1
was evident by immunofluorescence analysis (), FACS analysis (), and Western blot (). Furthermore, most of the cells that accumulated p27Kip1
in the nucleus had a corresponding loss of Ki-67 staining ().
Figure 4 UBE2E3 knockdown causes a dramatic elevation of p27Kip1 levels. (A) Representative FACS data of cells treated with siCON siRNA (left) or UBE2E3-specific siRNA duplexes 1 to 4 (Si2E3, right). Two days post-siRNA treatment, cells were colabeled with an (more ...)
The specificity of the p27Kip1 elevation was confirmed by rescue experiments. We first generated a rescue plasmid containing the cDNA sequence for wild-type, untagged, UBE2E3 but harboring silent point mutations such that the mRNA produced would be impervious to UBE2E3-specific siRNA silencing. We used anti-2E3 Western blotting to validate that this plasmid expressed untagged UBE2E3 () and importantly, that this expression was refractory to siRNA-mediated silencing (). Control cells were transfected with a construct encoding RFP-H2B. We observed that transfection of RPE-1 cells with the rescue plasmid resulted in an increase in UBE2E3 levels (). UBE2E3 overexpression also led to the production of both a faster-migrating species (e.g., ) and a high-molecular-weight smear that likely represents auto-ubiquitylated enzyme (e.g., ). In cells cotreated with UBE2E3-specific siRNA, UBE2E3 expression from the rescue plasmid was readily apparent, as were the faster-migrating band, and the high-molecular-weight species ().
Figure 5 The elevation of p27Kip1 levels by UBE2E3 depletion can be rescued by reintroduction of the enzyme. (A) α-2E3 Western blots of lysates derived from RPE-1 cells transfected with plasmids expressing RFP-H2B (Mock) (lane 1) or untagged UBE2E3 (lane (more ...)
We next performed the rescue experiment by transfecting RPE-1 cells with either the rescue plasmid or a control plasmid encoding RFP-H2B. The following day, the cells were treated with siRNA, and 3 days later, they were fixed, permeabilized, and processed for anti-p27Kip1 immunofluorescence (). A moderate decrease in p27Kip1 levels was observed in the UBE2E3-rescued cells compared with the mock-rescued cells (). Quantitation of nuclear p27Kip1 levels confirmed that expression of siRNA-impervious UBE2E3 mRNA successfully lowered p27Kip1 levels (). We attribute the intermediate level of rescue to inefficient transfection and/or heterogenous expression from the rescue plasmid among the population of cells. Collectively, these studies that UBE2E3 is essential for the proliferation of RPE-1 cells, and depletion of the enzyme induces a p27Kip1-mediated cell cycle exit.
One prediction of these tissue culture findings is that UBE2E3 promotes RPE cell proliferation during eye development but is then downregulated as the cells transition toward terminal differentiation. To test this prediction, we compared the expression profile of UBE2E3 in the developing and mature mouse RPE. As mentioned, the mouse counterpart of UBE2E3 is called UbcM2, and the proteins are 100% identical. We generated a unique mouse strain to analyze the in vivo expression profile of UbcM2. This 129/SvEv strain harbors one wild-type UbcM2 allele and one allele disrupted with a β
-gal-neo insertion under control of the endogenous UbcM2 promoter. This allele expresses the amino-terminal 86 residues of UbcM2 fused in-frame to β
-gal-neo. Thus, every cell that expresses UbcM2 expresses the β
-gal-neo fusion protein. In addition, both the β
-galactosidase and neomycin phosphotransferase II enzymes are functional in the context of the fusion protein (e.g., and Ref. 29
Figure 6 UbcM2, the mouse counterpart of UBE2E3, is downregulated during RPE maturation in the developing mouse eye. (A) Representative photomicrographs of X-Gal-labeled cryosections from E13 embryos (Aa, Ab) and P17 mice (Ac, Ad). (Ab, Ad) Wild-type samples; (more ...)
We used the β
-gal activity as a heterologous reporter to compare the expression of UbcM2 in proliferating RPE cells versus mature RPE cells. This method enabled us to bypass the inability of α
-2E3 to specifically detect nuclear enzyme in paraffin-embedded sections and cryosections (data not shown). Cryosections from E13 (representing developing RPE) and P17 (representing mature RPE) mice were prepared and stained in parallel with X-Gal as described in the Materials and Methods section. E13 was chosen as the early time point because RPE cells proliferate at this time, and robust p27Kip1
expression in the developing mouse eye is not observed until E18.30
After an overnight fixation in 10% formaldehyde, the sections were bleached24
to reduce the amount of melanin pigment in the RPE and choroid and thereby facilitate visualization of the X-Gal signal. Representative bright-field photomicrographs from each sample along with sections processed in parallel from wild-type littermates demonstrate the specificity of the reporter system (e.g., ). Quantification of the X-Gal spots per millimeter of RPE revealed that β
-gal-neo expression was on average 3.3-fold higher at E13 versus p17 (). These data demonstrate that in vivo, UbcM2 expression is transcriptionally downregulated during development as RPE cells mature and transition from a proliferative state to one of terminal differentiation. Notably, the X-Gal staining also revealed robust expression from the UbcM2 promoter in several regions of the adult mouse retina including the inner segments of the photoreceptors, the outer plexiform layer, and to a lesser extent, the outer and inner nuclear layers (). The function(s) of UbcM2 in the retina is currently under investigation.