Cloning of hRIPα and its isoforms.
Searches of the expressed sequence tag database identified the hRIPα protein as a human homologue of xRIPα, with the two proteins displaying similar domain structures (19
). hRIPα cDNA was obtained by RT-PCR using RNA extracted from Jurkat cells as a template and primers complementary to the 5′ and 3′ ends of the hRIPα open reading frame, which encodes the full-length hRIPα protein (Fig. ).
FIG. 1. hRIPα gene structure and alternative splicing. (A) Schematic diagram of the hRIPα gene and the alternative splicing that leads to its multiple isoforms. Coding exons are indicated by black boxes, as mapped on bacterial artificial chromosome (more ...)
RT-PCR analysis revealed that hRIPα yields seven splice isoforms, and various cell lines, including HeLa, U2OS, BJAB, Jurkat, and HEK293T, were employed to verify expression of the alternatively spliced mRNAs (Fig. ). Characterization of the splice isoforms of hRIPα was accomplished by cloning the various PCR products into a hemagglutinin (HA)-tagged eukaryotic expression vector (pcDNA3/HA) and obtaining their nucleotide sequences by standard methods. Seven isoforms were cloned, and nucleotide sequencing indicated that each was derived from a unique combination of hRIPα exons 1 through 7 (Fig. ).
Any one of the seven clones can potentially encode the polypeptide, and further analysis was necessary. The present work yielded seven different cDNA clones, but two of these (hRIPδ1a and hRIPδ1b) were found to encode the same polypeptide (hRIPδ1). Thus, a total of six polypeptides were produced through alternative splicing of the hRIPα gene (Fig. ). The longest gene product was the hRIPα protein, and the smaller polypeptides were named hRIPβ, hRIPγ, hRIPδ1, hRIPδ2, and hRIPδ3 in order of decreasing length. While hRIPα and hRIPβ are terminated by the same codon, the termination codons of the remaining clones are produced by a frameshift within their cDNA sequences. hRIPδ1, hRIPδ2, and hRIPδ3 have been grouped into the hRIPδ family, as they differ by only 2 or 3 amino acids while sharing 104 amino acids (Fig. ).
hRIPβ localizes to the PML nuclear body.
In order to determine which of the splice variants indeed encode the protein of interest, hRIPα and its various splice isoforms were expressed in rabbit reticulocytes in the presence of [35S]methionine. PAGE revealed that hRIPα and all of its isoforms could be translated into a protein of the appropriate size in vitro (Fig. ). Their expression in the eukaryotic cell was verified by transfection of plasmids encoding hRIPα and its isoforms into HEK293T cells and subsequent immunoblot analysis with anti-HA antibody. While hRIPα and hRIPβ proteins were readily detected, the expression of other isoforms was low, indicating that hRIPα and hRIPβ are the major splice isoforms in vivo (Fig. ). Frequently throughout the experiment reported here, hRIPβ expression resulted in a shifted band at around 45 kDa that will be discussed further in the next section.
FIG. 2. Expression analysis of hRIPα and its isoforms and differential localization of hRIPα and hRIPβ. (A) In vitro translation of hRIPα and its isoforms. hRIPα and its isoforms were translated in vitro using [35S]methionine (more ...)
Endogenous expression of hRIPα and hRIPβ was demonstrated by generating a rabbit polyclonal antibody against bacterially expressed GST-hRIPα recombinant protein that contained full-length hRIPα and was named hRIP antibody. Additionally, an hRIPα-specific antibody was generated using hRIPα-specific exon 6 domain to recognize the endogenous hRIPα expression and was named hRIPAS (alpha-specific) antibody. A GST-hRIPα deletion mutant (amino acid 107 to 219) was used to immunize rabbits, and the antibody, which recognized GST-hRIPβ recombinant protein, was removed to obtain specificity to hRIPα as described in Materials and Methods. Immunoblotting with the hRIP antibody revealed that it could recognize the double bands near 30 kDa as well as the 45-kDa band. The larger band is consistent with the shifted band of HA-hRIPβ, while the two smaller bands approximate HA-hRIPα and HA-hRIPβ. Finally, immunoblotting with the hRIPAS antibody demonstrated that the upper of the smaller two bands is hRIPα. These findings strongly support hRIPα and hRIPβ as the major translation products of the hRIPα gene (Fig. ).
Since hRIPα and hRIPβ are predominantly expressed in eukaryotic cells, the initial focus here was on the subcellular localization of these two isoforms. Plasmids encoding HA-hRIPα and HA-hRIPβ were transiently expressed in HEK293T cells, which were then immunostained with anti-hRIP antibody. Confocal microscopy revealed hRIPα to be localized primarily in the cytoplasm but also detectable in the nucleus. By contrast, the majority of hRIPβ was localized to the nucleus with a dot-like staining pattern (Fig. ). These results were confirmed in an analogous experiment with COS cells and anti-HA antibody (Fig. ). The distinct subcellular localizations of hRIPα and hRIPβ were corroborated by transient expression of hRIPα and hRIPβ in cells that were subjected to cellular fractionation to give cytoplasmic and nuclear fractions. While the majority of hRIPα proteins were detected in the cytoplasmic fraction, hRIPβ proteins were mostly found in the nuclear fraction (Fig. ). Taken together, these results indicate that hRIPα and hRIPβ indeed display distinct subcellular localization patterns.
After observing the dot-like localization of hRIPβ in the nucleus, investigation shifted to further characterizing these dot-like structures. Previous studies had documented that RPA, the binding partner of hRIPα, was often found in PML nuclear bodies (1
), and because of this finding it was postulated here that the observed nuclear spots may have indeed been PML nuclear bodies. This hypothesis was tested by transfecting plasmids encoding hRIPα and hRIPβ into HEK293T cells and then double immunostaining with anti-hRIP antibody (rabbit, green) and anti-PML antibody (mouse, red). Confocal microscopy revealed that more than 90% of hRIPβ foci were completely associated with PML spots in the transiently transfected cells, indicating that hRIPβ localized to the PML nuclear body (Fig. ).
Sumoylation of hRIPβ.
When hRIPβ was transiently expressed in HEK293T cells, there was repeated detection of a more slowly migrating form of hRIPβ (slower by ~20 kDa). This band was also detected by anti-hRIP blotting using endogenous proteins (Fig. ). Because many proteins in the PML nuclear body are modified by sumoylation (28
), it was assumed that the larger band was the sumoylated form of hRIPβ. This hypothesis was investigated by determining whether hRIPβ was in fact sumoylated in vivo.
Transfections were performed that paired plasmids encoding HA-hRIPα and HA-hRIPβ with plasmids for FLAG-SUMO-1 and thus revealed if conjugation was taking place. Figure shows that the more slowly migrating form of hRIPβ was detected by both anti-HA and anti-FLAG antibodies but was not detected with anti-FLAG antibody in the absence of FLAG-SUMO-1 expression. These findings indicate that the slower-migrating hRIPβ band is the sumoylation product of the protein.
FIG. 3. Sumoylation of hRIPβ and generation of SUMO-deficient hRIPβ mutants. (A) Sumoylation of hRIPβ. HEK293T cells were transiently transfected with plasmids encoding hRIPα or hRIPβ in combination with a plasmid encoding (more ...)
So as to provide further evidence for the sumoylation of hRIPβ in vivo, sumoylated hRIPβ was immunopurified. SUMO protease is capable of readily cleaving the isopeptide bond between SUMO and the target protein, so the cell lysate was boiled to inactivate SUMO protease (32
). The denatured cell lysates were incubated with anti-FLAG antibody in order to purify proteins conjugated to FLAG-SUMO-1, and the resulting precipitates were probed with anti-HA antibody for detection of HA-hRIPβ. The band corresponding to FLAG-SUMO-1-conjugated HA-hRIPβ was purified and positively identified by anti-HA antibody (Fig. ). hRIPβ was thus shown to be a substrate for sumoylation.
The obvious extension of showing that hRIPβ can be sumoylated was to determine the significance of such a modification to the protein. To this end, a SUMO-deficient mutant of hRIPβ was generated: the lysine residue of the sumoylation consensus sequence (IKQE) was replaced with asparagine (30
). Yet sumoylation of hRIPβ was not completely diminished by the K103N mutation (hRIPβ KM-1) (Fig. ), and the other lysine residues were replaced by arginine so as to generate additional mutants. As shown in Fig. , sumoylation of hRIPβ 2KM-2 (carrying both the K103N and K121R mutations) was significantly reduced, and the sumoylated form was completely lost for hRIPβ 4KM, modified by the K103N and K114,121,142R mutations. This strongly implicates the multiple lysine residues as the acceptor site of sumoylation (Fig. ).
Establishment of the sumoylation-deficient mutants of hRIPβ led to an examination of the role of hRIPβ sumoylation. Plasmids encoding hRIPα, hRIPβ, and hRIPβ 4KM were transiently transfected with GFP-SUMO-1, whose expression showed dense spots, which indicate PML nuclear bodies (Fig. , panel iv). More than 90% of hRIPβ foci showed complete association with GFP-SUMO-1 spots, while none of the hRIPα was colocalized with GFP-SUMO-1 (Fig. , panels i and ii). In contrast, 70% of hRIPβ 4KM exhibited spots with no association or only adjacent localization with GFP-SUMO-1 (Fig. , panel iii). The remaining hRIPβ 4KM was diffusely dispersed in the nucleus. These results suggest that hRIPβ must be sumoylated in order to localize to the PML nuclear body.
Interaction between RPA and hRIPα splice isoforms.
Jullien et al. (19
) reported that RPA p70 is the binding partner of hRIPα and that the acidic-residue-rich domain of hRIPα is the binding region for RPA p70. Because both hRIPα and hRIPβ contain this acidic domain, we examined whether hRIPβ could interact with the RPA p70 protein. The interaction between RPA and hRIPβ was probed by transfecting HEK293T cells with plasmids encoding FLAG-tagged RPA p70 (FLAG-RPA) in the presence or absence of GST-hRIPα and GST-hRIPβ. Forty-eight hours after transfection, GST, GST-hRIPα, and GST-hRIPβ were precipitated with glutathione beads. Bead-bound FLAG-RPA proteins were probed with anti-FLAG antibodies. A binding assay revealed that both hRIPα and hRIPβ interacted with the RPA protein in vivo (Fig. ). Because RPA p70 is one subunit of the RPA complex, it was demonstrated that the transiently expressed FLAG-RPA p70 was associated with endogenous RPA p32 in vivo (Fig. ). A coimmunoprecipitation assay with endogenous proteins provided yet more evidence for the interaction between RPA and hRIPα/hRIPβ. This coimmunoprecipitation assay with HEK293T cells also showed interactions of RPA with endogenous hRIPα and hRIPβ (Fig. ).
FIG. 4. Interaction between RPA and hRIPβ. (A) hRIPα and hRIPβ interact with RPA in HEK293T cells. Cells were transiently transfected with a plasmid encoding FLAG-RPA in combination with GST, GST-hRIPα, or GST-hRIPβ. Whole-cell (more ...)
In order to elucidate the interactions between RPA and the many splice isoforms of hRIPα, a GST pull-down assay was performed. GST-RPA p70 recombinant protein was produced in bacteria (Fig. ), and the in vitro binding assays used in vitro-translated forms of hRIPα, hRIPβ, and hRIPδ3. While the last of these, hRIPδ3, interacted with the RPA protein, the hRIPα mutant (amino acids 1 to 84) devoid of the acidic domain lacked binding affinity to RPA (Fig. ), as shown previously by (19
). An hRIPδ3 mutant (amino acids 45 to 106) lacking the basic domain required for interaction with importin β was also constructed. While the hRIPδ3(45-106) mutant did not interact with importin β in vitro (data not shown), an interaction with RPA was identified that was slightly weaker than that of normal hRIPα and its splicing variants (Fig. ). These results indicate that the splice isoforms of hRIPα, as well as hRIPα, interact with RPA.
Nuclear export signal sequence of hRIPα.
Based on an in vitro transport assay, Jullien et al. (19
) reported that xRIPα transports RPA into the nucleus, but the detailed mechanism of the process was not known at the time. Because hRIPα localizes primarily to the cytoplasm, it is supposed that hRIPα shuttles between the nucleus and the cytoplasm and thereby transports RPA into the nucleus. This hypothesis was supported by identifying the region of hRIPα required for nuclear export. Because hRIPα and hRIPβ share the majority of their peptide sequences yet display different subcellular localizations, it was supposed that the additional hRIPα-specific domain (amino acids 164 to 210) induced nuclear export of that protein (Fig. ). To verify this hypothesis, several deletion mutants that displayed differential localization were generated (Fig. ).
FIG. 5. hRIPα contains the specific domain for cytoplasmic localization. (A) Schematic diagram of hRIPα, its isoforms, and deletion mutants. Numbers correspond to the amino acid sequence. Note the presence or absence of the indicated region (amino (more ...)
Confocal microscopy data revealed that hRIPβ, hRIPγ, and hRIPδ3 generally localized to the nucleus in a dot-like pattern (Fig. ). Because hRIPβ, hRIPγ, and hRIPδ3 lack the hRIPα-specific domain (amino acids 164 to 210), however, one can conclude that the hRIPα-specific domain is required for nuclear export. Moreover, two hRIPα deletion mutants (amino acids 1 to 180 and 1 to 200) localized to the cytoplasm (Fig. ), indicating that the 17-amino-acid region from amino acid 164 to 180 was sufficient for nuclear export of hRIPα.
Interaction with importin β is also important for nuclear import, as illustrated by an hRIPδ3 mutant that does not interact with importin β and localizes to the cytoplasm. On the other hand, an hRIPα(1-84) mutant that does not interact with RPA localizes to both the nucleus and cytoplasm. The above results indicate that hRIPα indeed harbors the specific domain (amino acids 164 to 180) required for its nuclear export.
hRIPβ targets RPA to the PML nuclear body.
Previous studies documented that RPA protein was mainly or partially localized to the PML nuclear body and that colocalization with the PML nuclear body was dependent on cell type (1
). Little is known, however, about the transport of RPA into the PML nuclear body. As hRIPβ is associated with RPA and also localizes in the PML nuclear body, we postulated that hRIPβ was the carrier molecule for the targeting of RPA to the PML nuclear body.
To assess whether hRIPβ indeed targets RPA to the PML nuclear body, RPA was transiently expressed in HEK293T cells with either blank vector or a plasmid encoding hRIP isoforms and mutants. After 24 h, the transfected cells were fixed and double immunostained with anti-hRIP antibody (rabbit, green) and anti-FLAG antibody (mouse, red). While ectopic expression of FLAG-RPA alone induced accumulation of FLAG-RPA protein in the cytoplasm, expression of hRIPα, hRIPβ, and hRIPδ3 led to the transport of RPA protein into the nucleus (Fig. ).
FIG. 6. hRIPβ transports RPA into the PML nuclear body. HEK293T cells were transfected with a plasmid encoding FLAG-RPA in combination with either blank vector or hRIP constructs. After 24 h, the cells were fixed and stained with anti-hRIP antibody (green) (more ...)
The nuclear distribution pattern of RPA with hRIPβ differs from the pattern of RPA with hRIPα. While RPA localizes throughout the entire nucleus when hRIPα is expressed, hRIPβ expression leads to a dot-like staining pattern of RPA. Previous microscopy data suggest that these dots are PML nuclear bodies. Moreover, hRIPβ was tightly associated with RPA, and hRIPα did not colocalize with RPA. While a fraction of the hRIPβ 4KM mutant colocalized with RPA, the level of this colocalization was weaker than that for wild-type hRIPβ. In the case of the hRIPα(1-84) mutant, which was devoid of the RPA binding region, localization was limited to the cytoplasm, indicating that the interaction between RPA and hRIPα or its isoforms is required for the transport of RPA into the nucleus.
hRIPβ is desumoylated upon UV irradiation and during S phase of the cell cycle.
Negorev and Maul (28
) postulated that the PML nuclear body acts as a nuclear depot and can recruit specific proteins and release them upon external stress. Furthermore, regulated recruitment into the PML nuclear body, as well as controlled release, is closely related to sumoylation of the proteins. Because hRIPβ can transport RPA into the PML nuclear body and because this process is dependent on the sumoylation of hRIPβ, it is supposed that hRIPβ recruits RPA into the PML nuclear body and releases RPA upon external stress. UV irradiation was used to determine if the hRIPβ protein would be desumoylated by an external stressor.
HA-hRIPβ was transiently expressed in HEK293T cells, and the cells were either left untreated or irradiated with 50 J/m2 of UV light. After UV irradiation, the cells were harvested and subjected to direct lysis. The sumoylation status of hRIPβ was examined by immunoblot analysis with anti-HA antibody. As shown in Fig. , the sumoylated hRIPβ band was reduced to less than half that for untreated cells at 1 h postirradiation. By 2 h postirradiation, sumoylated hRIPβ was hardly detected, although the amount of unmodified hRIPβ had not been significantly altered (Fig. , panel i). A similar experiment with the endogenous proteins was performed to provide more evidence, and the results with the endogenous hRIPβ were identical to those with the transiently expressed hRIPβ (Fig. , panel ii). These results indicate that UV light desumoylates hRIPβ in vivo.
FIG. 7. Sumoylation of hRIPβ is regulated by UV irradiation and the normal cell cycle. (A) Panel i, UV irradiation desumoylates hRIPβ. HA-hRIPβ was transiently expressed in HEK293T cells. After 24 h, the cells were either left untreated (more ...)
Recently, the SUMO isopeptidase SENP1 was identified (14
). SENP1 is responsible for the desumoylation of the proteins in the PML nuclear body upon external stress (27
). Because hRIPβ is desumoylated by UV irradiation, it is supposed that SENP1 removes SUMO from hRIPβ. This hypothesis was tested by expressing HA-hRIPβ in HEK293T cells in the presence or absence of His-SENP1. Figure , panel i, shows that transient expression of SENP1 results in a dramatic reduction of sumoylated hRIPβ. The same results were obtained with the endogenous hRIPβ (Fig. , panel ii). To further confirm the effect of SENP1 on regulation of hRIPβ desumoylation, a SENP1 siRNA plasmid was used to silence endogenous SENP1 in HEK293T cells. Cheng et al. (6
) reported that SENP1 siRNA treatment decreases the endogenous SENP1 mRNA. As expected, the desumoylation of hRIPβ upon UV irradiation was decreased in SENP1 siRNA-treated cells (Fig. , panels iii and iv). Consequently, SENP1 is responsible for the desumoylation of hRIPβ.
Next, the localization of hRIPβ and RPA upon UV irradiation was examined. hRIPβ and FLAG-RPA were expressed in HEK293T cells, and these cells were either left untreated or irradiated with 50 J/m2 of UV light. At 2 h postirradiation, the cells were fixed and reacted with anti-hRIP antibody (green) and anti-FLAG antibody (red). While hRIPβ showed obvious colocalization with RPA without UV treatment (Fig. , upper panels), UV irradiation led to the dissociation of RPA from hRIPβ (Fig. , lower panels). hRIPβ still showed dot-like patterns in the nucleus, but the spots were enlarged and the level of colocalization with RPA was much weaker than it was without UV treatment. RPA also exhibited smaller nuclear foci, which were believed to be the sites of DNA damage and repair as had been previously reported (1
). These results indicate that UV light disrupts the interaction between hRIPβ and RPA, with this process accompanied by the desumoylation of hRIPβ.
Further demonstration of the localization change of RPA with hRIPβ upon treatment with UV light was accomplished through immunohistochemistry with endogenous RPA and hRIPβ protein in U2OS cells. Background interference was reduced by using the more sensitive anti-RPA p32 antibody and the purified anti-hRIP antibody to detect the endogenous protein. It should be recognized that all of the hRIPβ amino acid sequences are also included in hRIPα sequences. This redundancy contributed to difficulties when attempting to immunostain hRIPβ with the endogenous proteins. Purified anti-hRIP antibody detects the splice isoforms of hRIPα as well as hRIPβ. For this reason, anti-hRIPAS antibody was used to stain endogenous hRIPα as a control. As shown in Fig. , anti-hRIP antibody reacts with the spots in the nucleus, and anti-RPA also reacts with the same spots. As a control, anti-hRIPAS antibody was used to detect the endogenous hRIPα, and anti-hRIPAS antibody does not react with the spots whereas anti-RPA antibody does. These data strongly support the supposition that hRIPβ transports RPA into the PML nuclear body.
In the same way, the localization of endogenous hRIPβ and RPA upon UV irradiation was examined. After UV irradiation, endogenous RPA was translocated to small and abundant spots (Fig. ). While a fraction of RPA spots were colocalized with hRIP spots, the rest of them were not colocalized with hRIP spots, indicating that the level of colocalization of RPA with hRIPβ was reduced upon UV irradiation. Taken together with the transient-expression data, these results indicate that UV light induces the dissociation of RPA from hRIPβ.
Finally, changes in hRIPβ sumoylation status were tracked throughout the cell cycle. Jurkat cells were synchronized using the thymidine double-block method (17
). After thymidine removal, the cells were harvested at the indicated times and subjected to cell cycle analysis (Fig. ). Immunoblotting with anti-hRIP antibody showed that sumoylation of endogenous hRIPβ was lowered during early and full S phase (4 h and 8 h after thymidine removal, respectively) and that sumoylation was increased during G2
/M phase (Fig. ). These findings show that sumoylation of hRIPβ varies during the cell cycle.