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Molecular Endocrinology, doi:10.1210/me.2006-0414
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Molecular Endocrinology 21 (3): 686-699
Copyright © 2007 by The Endocrine Society

Pus3p- and Pus1p-Dependent Pseudouridylation of Steroid Receptor RNA Activator Controls a Functional Switch that Regulates Nuclear Receptor Signaling

Xiansi Zhao, Jeffrey R. Patton, Sajal K. Ghosh, Nathan Fischel-Ghodsian, Ling Shen and Remco A. Spanjaard

Departments of Otolaryngology and Biochemistry (X.Z., L.S., R.A.S.), Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118; Department of Pathology and Microbiology (J.R.P.), University of South Carolina School of Medicine, Columbia, South Carolina 29208; Department of Medicine (S.K.G.), Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Pediatrics and Medical Genetics Institute (N.F.-G.), Cedars-Sinai Medical Center, University of California, Los Angeles, School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Remco A. Spanjaard, Ph.D., Cancer Research Center, Boston University School of Medicine, 715 Albany Street R903, Boston, Massachusetts 02118. E-mail: rspan{at}bu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It was previously shown that mouse Pus1p (mPus1p), a pseudouridine synthase (PUS) known to modify certain transfer RNAs (tRNAs), can also bind with nuclear receptors (NRs) and function as a coactivator through pseudouridylation and likely activation of an RNA coactivator called steroid receptor RNA activator (SRA). Use of cell extract devoid of human Pus1p activity derived from patients with mitochondrial myopathy and sideroblastic anemia, however, still showed SRA-modifying activity suggesting that other PUS(s) can also target this coactivator. Here, we show that related mPus3p, which has a different tRNA specificity than mPus1p, also serves as a NR coactivator. However, in contrast to mPus1p, it does not stimulate sex steroid receptor activity, which is likely due to lack of binding to this class of NRs. As expected from their tRNA activities, in vitro pseudouridylation assays show that mPus3p and mPus1p modify different positions in SRA, although some may be commonly targeted. Interestingly, the order in which these enzymes modify SRA determines the total number of pseudouridines. mPus3p and SRA are mainly cytoplasmic; however, mPus3p and SRA are also localized in distinct nuclear subcompartments. Finally, we identified an in vivo modified position in SRA, U206, which is likely a common target for both mPus1p and mPus3p. When U206 is mutated to A, SRA becomes hyperpseudouridylated in vitro, and it acquires dominant-negative activity in vivo. Thus, Pus1p- and Pus3p-dependent pseudouridylation of SRA is a highly complex posttranscriptional mechanism that controls a coactivator-corepressor switch in SRA with major consequences for NR signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS (NRs) belong to a superfamily of conserved transcriptional modulators that control vertebrate development, cell growth, and homeostasis in metazoa (1, 2), but they also play a role in tumor development (3, 4, 5). Dissection of the molecular mechanism of NR-mediated gene activation is, therefore, of biological importance. It is known that, in the absence of ligand, NR homodimer or heterodimer-directed transcription is prevented because they are associated with a multiprotein corepressor complex with histone deacetylase activity. When ligand is present, a conformational change in the ligand-binding domain (LBD) results in release of corepressors and recruitment of a coactivator complex with histone acetyltransferase and methyltransferase activities and subsequent acetylation or methylation of chromatin, respectively (6, 7, 8, 9, 10). The list of identified coactivators essentially comprises two major categories: the p160/SRC-1 family, which contains SRC-1/NcoA-1/ERAP-160, TIF-2/GRIP-1/NCoA-2/SRC-2, and AIB1/p/CIP/ACTR/RAC3/SRC-3/TRAM-1, and second, the CBP/p300 family of transcriptional cointegrators; however, many coactivators fall outside these groups (11).

One of the most surprising coactivators is called steroid receptor RNA activator (SRA) (12). SRA functions as an RNA molecule, although certain 5' splice variants that encode a translated protein have also been reported (13, 14). SRA is suspected of involvement in breast carcinogenesis via aberrant overstimulation of estrogen receptor (ER) activity (15, 16, 17), and thus analysis of its molecular action is highly relevant. SRA exists in a stable ribonucleoprotein complex with SRC-1 with class I and II NRs at the LBD, and only associates with the N terminus of class I NRs in the absence of SRC-1 (12). However, recently SRA was also shown to stimulate the activity of nonsteroid receptors such as the thyroid hormone receptor (TR) and retinoic acid receptor (RAR) (18, 19). Additionally, SRA associates with DEAD-box protein p72/p68 in an ER{alpha}-specific coactivator complex (20) and NR corepressors SHARP (21) and SLIRP (22). The SHARP/SRA association is thought to at least partially modulate ER transactivation through sequestration of SRA, whereas SLIRP binding to SRA enhances NCoR promoter recruitment. Thus, SRA may serve as a scaffold for proteins involved in repression and activation of NR-dependent signaling, and many others still await identification (22). Interestingly, SRA was recently also found to be a coactivator of muscle cell differentiation factor MyoD (23), and it, therefore, appears that SRA may have broader biological activity than initially thought.

Computer-assisted modeling suggests that SRA adopts a highly complex secondary structure of the essential core region that consists of exons 2–5 (15). Whereas stem-loop structure (STR)7 was identified as being important for SRA function (15, 22), the specific contributions of other higher order RNA structures to SRA activity remain largely unknown.

It was recently established that SRA requires a novel type of posttranscriptional modification by a coactivator named mouse Pus1p (mPus1p) to function in NR-dependent transactivation (19). mPus1p is a member of the pseudouridine synthase (PUS) family which isomerizes uridine (U) to pseudouridine ({Psi}) in noncoding RNAs such as transfer RNA (tRNA), ribosomal RNA, small nuclear RNA, and possibly small nucleolar RNA (24, 25). Pus1p, together with Pus3p and bacterial truA, form the small truA subfamily whose products modify specific positions in many tRNAs (26, 27). {Psi} has an additional iminoproton that helps stabilize base stacking (28) and hydrogen bonding (29, 30, 31), which in turn helps establish specific intramolecular and intermolecular interactions within the RNA, or between RNA-RNA and RNA-protein(s), respectively (25). The presence of {Psi} is required for correct tRNA codon reading (32) and spliceosome assembly (33). The physiological importance of appropriate PUS activity is illustrated by disorders such as dyskeratosis congenita (34) and associated cancer (35, 36), which is caused by a point mutation in telomerase component Dyskerin, a PUS in the truB family. Furthermore, an inactivating mutation in human Pus1p (hPus1p) causes mitochondrial myopathy and sideroblastic anemia (MLASA) (37, 38, 39), and it has been suggested that other abnormalities in these patients, such as facial dysmorphisms, may be the consequence of defective hSRA-NR signaling (38).

Here, we show that mPus3p can also serve as NR coactivator except that, unlike mPus1p, it does not enhance sex steroid receptor activity. mPus1p and mPus3p modify different positions in SRA, although a few positions may be commonly targeted. Interestingly, the order in which SRA is modified by mPus1p and mPus3p determines how many positions are pseudouridylated. Finally, we identified the first in vivo-modified U in SRA, which may be a common target for mPus1p and mPus3p. Mutagenesis of this U to A allows for the hyperpseudouridylation of SRA, and switches SRA from a coactivator to a molecule with dominant-negative activity. These results show that pseudouridylation by mPus1p and mPus3p is a highly complex posttranscriptional mechanism that controls a coactivator-corepressor switch in SRA with major consequences for NR signaling.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MLASA Patient Cell Extract Has SRA-Modifying Activity
MLASA is a rare, debilitating autosomal recessive disorder of oxidative phosphorylation and iron metabolism that is caused by an inactivating missense mutation in NR coactivator hPus1p (37, 38, 39). Because MLASA patient-derived cells are deficient in hPus1p activity, they were used to test whether hPus1p is the only PUS that can modify SRA, or whether there are other PUSs that recognize it as a substrate. This is an important question, because these putative PUSs may also be NR coactivator(s). To address this issue, nuclear extracts from immortalized, lymphoblastoid cells from a homozygous MLASA patient, and from a normal sibling control (39), were incubated with in vitro-synthesized hSRA to determine pseudouridylation activity in a standard assay. As shown in Fig. 1Go, MLASA extract has somewhat less, but still significant dose-dependent hSRA-modifying activity compared with control extract, suggesting the activity of an undetermined PUS(s).


Figure 1
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  		Fig. 1. MLASA Nuclear Extract Still Contains hSRA-Modifying Activity

In vitro pseudouridylation assay showing that hPus1p-deficient nuclear extract derived from lymphoblastoid cells from a MLASA patient still has significant hSRA-modifying activity compared with a sibling control, suggesting that there is another PUS that targets hSRA. Indicated amounts of nuclear extracts were incubated with in vitro-generated hSRA, and moles of {Psi}/moles of hSRA was determined after 1 h.

 
mPus3p Is a Novel NR Coactivator except for Sex Steroid Receptors
Although the PUS family is extensive and poorly characterized in mammalian cells, a likely candidate for this activity is hPus3p. This related enzyme has a different tRNA and position specificity than hPus1p (27), but it is the only other mammalian truA family member, and it is known to be expressed in these MLASA cells (39). Therefore, we decided to focus on the potential coactivator function of mPus3p. To test this hypothesis, S91 cells that contain endogenous mRAR{gamma} activity were transfected with Luciferase (Luc) reporter plasmid pRARE3Luc together with mPus3p expression vector, in the absence or presence of RAR{gamma} agonist CD666 (19). As shown in Fig. 2AGo, a mPus3p dose-dependent enhancement of mRAR{gamma}-mediated transactivation was observed in the presence of ligand. A reporter plasmid without a retinoic acid response element (RARE) ({Delta}RARE) was unresponsive to mPus3p overexpression and neither the NR-independent RSV nor the E2F promoters stimulated by mPus3p (Fig. 2BGo), showing that the effects required DNA binding by mRAR{gamma} and did not generally affect gene expression. Moreover, mPus3p coactivator results were confirmed in a reciprocal RT-PCR analysis of expression of endogenous RAR-target gene mRARß. S91cells that were depleted of mPus3p by short interfering RNA (siRNA) showed significantly less induction of mRARß in the presence of CD666 than control siRNA-transfected cells (Fig. 2CGo). These combined results establish that mPus3p acts as coactivator of liganded mRAR{gamma}.


Figure 2
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  		Fig. 2. mPus3p Is a Novel, Specific Coactivator of NR-Dependent Transactivation except for Sex Steroid Receptors

A, Ligand- and dose-dependent effect of mPus3p-enhanced mRAR{gamma}-mediated transcriptional activation. S91 cells were transfected with RARE3Luc and different doses of mPus3p plasmids, and ligand (1 µM CD666) as indicated. Luc assays were performed with the cell extracts and, after normalization, expressed as arbitrary Luc units. * and **, P ≤ 0.005 (by Student’s t test analysis). B, mPus3p does not stimulate mRAR{gamma} activity in the absence of an RARE in {Delta}RARELuc reporter (left), nor does it nonspecifically increase RSVLuc or E2FLuc activity (right). C, RT-PCR analysis showing that mRAR{gamma}-dependent induction of endogenous mRARß via treatment with CD666 (16 h; 1 µM) is inhibited in cells that were treated with siRNA-Pus3p, but not with siRNA-control as indicated. ß-Actin serves as RNA control. D, Ligand-dependent, mPus3p-enhanced transactivation of endogenous VDR, and transfected TRß and GR but not sex steroid receptors ER{alpha}, PRß, and AR-transactivated Luc reporter plasmids. *, P ≤ 0.03, P ≤ 0.0004, P ≤ 0.004, respectively (by Student’s t test analysis). S91 cells were used except when indicated otherwise. E, Coimmunoprecipitation experiment showing that immunoprecipitated, transfected His-tagged mPus3p with anti-His antibodies specifically binds mRAR{gamma} but not hER{alpha} from S91 and MCF-7 cell lysates, respectively. Immunoblot with the indicated antibodies. F, In vitro-generated [35S]mPus3p specifically binds with GST-mRAR{gamma}-DBD but not with a GST-mRAR{gamma}-LBD fragment or GST control, suggesting that mPus3p binds with the DBD.

 
Next, we established the effects of mPus3p on transactivation by other representative class I and II receptors. S91 cells were transfected with appropriate reporter plasmids to determine activation by liganded, endogenous vitamin D3 receptor (VDR) or cotransfected TRß, glucocorticoid receptor (GR), ER{alpha}, progesterone receptor (PR)ß, or androgen receptor (AR). As shown in Fig. 2DGo, mPus3p significantly enhanced transactivation of all NRs except that, in contrast to mPus1p (19), mPus3p failed to increase activity of sex steroid receptors ER{alpha}, PRß, and AR. This was confirmed for endogenous ER{alpha} and AR in MCF-7 and LNCaP cells, respectively (data not shown). Because NR binding is critical for the coactivator activity of mPus1p (19), we theorized that mPus3p can bind to mRAR{gamma} but not to sex steroid receptors such as ER{alpha}. To test this hypothesis, S91 and MCF-7 cells were transfected with His-tagged mPus3p expression vector in the presence of CD666 and 17ß-estradiol (E2), respectively, and after 24 h, mPus3p was immunoprecipitated by anti-His or IgG control antibodies. We then established whether mRAR{gamma} from S91 cell lysate and hER{alpha} from MCF-7 cell lysate grown under standard culturing conditions was associated with immunoprecipitated His-mPus3p by immunoblotting. Our results show that, consistent with our transfection data, mRAR{gamma} was associated with His-mPus3p, whereas no detectable binding to hER{alpha} was observed (Fig. 2EGo), thus providing a plausible mechanism for the observed lack of mPus3p-dependent stimulation of sex steroid receptors. To confirm and expand upon this result, [35S]mPus3p was generated in rabbit reticulocyte lysate and incubated with affinity columns loaded with bacterially expressed glutathione S-transferase (GST)-tagged mRAR{gamma}-DNA-binding domain (DBD), GST-mRAR{gamma}-LBD, and GST alone. As shown in Fig. 2FGo, mPus3p specifically bound the mRAR{gamma}-DBD fragment, the same region that also binds with mPus1p (19).

mPus3p and mPus1p Pseudouridylate SRA in Different but Possibly Some Common Positions: The Order in which SRA Is Modified Determines the Total Number of {Psi}s
mPus1p-mediated pseudouridylation of SRA is critical for its coactivator activity (19), and it is conceivable that mPus3p uses a similar mechanism. To test this hypothesis, we incubated in vitro-generated hSRA and mSRA for various periods with mPus3p and mPus1p, and determined the total number of {Psi}s/hSRA in a standard pseudouridylation assay. Figure 3Go shows that mPus1p generated seven {Psi}s in hSRA and eight {Psi}s in mSRA, respectively. Consistent with our hypothesis, mPus3p also modifies SRA, generating six {Psi}s in hSRA and four {Psi}s in mSRA (Fig. 3Go). Modification kinetics of both enzymes was very similar, with saturation of modification starting at about 90 min and reaching a plateau after 180-min incubation, suggesting that available sites were completely isomerized after this time frame, as seen before (19). Both enzymes are present in excess, ensuring complete substrate modification as confirmed by the lack of significantly increased modification levels after addition of the same enzyme after 90-min incubation time (Fig. 3Go). mPus1p and mPus3p displayed expected specific modification of tRNA substrate controls (data not shown).


Figure 3
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  		Fig. 3. mPus1p and mPus3p Pseudouridylate SRA in Different, but also Some Common Positions, Whereas the Total Number of Pseudouridylated Positions Depends on the Order in which SRA Is Modified

A, Time-dependent in vitro pseudouridylation assay of hSRA with mPus1p (left) and mPus3p (right) shows that, when enzymes are used individually, seven and six positions are maximally modified after 180 min, respectively. Enzymes are in excess because additionally added enzyme after 90 min does not significantly increase number of modified positions. When hSRA is sequentially modified first by mPus1p (90 min) and then by mPus3p, saturation of modification is reached after 270 min with 10 modified positions, suggesting that some positions (≤3) are common targets (left). However, this process is asymmetrical because when hSRA is first modified by mPus3p (90 min) and then by mPus1p, no significant additional modification takes place (right). Background activity of similar reactions performed with control ß-galactosidase was subtracted from all time points (data not shown). B, Similar experiment with similar results as in A, except that mSRA was used as template. mPus1p (left) and mPus3p (right) maximally modify eight and four positions, respectively, and addition of more enzyme only increases mPus3p-dependent modification to five positions after 180 min. Sequential modification by mPus1p and mPus3p increases pseudouridylation to at least nine positions after 180 min (left), whereas premodification by mPus3p again inhibits further modification by mPus1p (right).

 
These results suggested that mPus3p and mPus1p modify different positions, as may be expected from their tRNA substrate-dependent specificities, or alternatively, mPus3p may only modify a subset of the same positions that are modified by mPus1p, or a combination thereof. To resolve this issue, we performed sequential modification reactions by first incubating SRA for 90 min with mPus1p, followed by addition of mPus3p for another 90 min. As shown in Fig. 3Go, after 180 min, the total number of {Psi}s in hSRA reached eight in hSRA and nine in mSRA, respectively. Moreover, saturation had not yet occurred after 180 min, and after extended incubation to 270 min, saturation was reached and about 10 {Psi}s were generated in hSRA (Fig. 3AGo). This result suggested that mPus3p and mPus1p modify different positions; however, because the number is lower than additive if all mPus3p- and mPus1p-targeted positions were different (~13 {Psi}s), it is possible that a few (less than or equal to three) sites are common targets. We then performed the reciprocal experiment in which we first modified SRA for 90 min with mPus3p, followed by another 90-min incubation with mPus1p. The results, shown in Fig. 3Go, were surprising, because here additional mPus1p did not result in increased pseudouridylation compared with the levels reached by mPus3p alone, suggesting that mPus3p-pseudouridylated SRA is no longer recognized by mPus1p. Thus, overall modification of SRA by Pus1p and Pus3p is an asymmetric process that depends on the dynamic interactions between these two factors. Our results also show that the presence of {Psi}s can alter protein recognition, a relevant finding in light of the putative scaffolding function of SRA.

mPus3p and mSRA Functionally Cooperate and mPus3p Occupies the mRARß2 Promoter in a Ligand-Independent Manner In Vivo
To verify that the interaction between mPus3p and SRA is important for NR-dependent transactivation, S91 cells were transfected with RARE3Luc reporter, and mPus3p and/or mSRA expression vectors. As shown in Fig. 4AGo, mPus3p again enhanced mRAR{gamma}-mediated transactivation, whereas mSRA alone had little effect, as reported earlier (19). However, when mPus3p and mSRA vectors were cotransfected, significant cooperativity was observed, showing that mSRA functionally interacts with mPus3p. Similar results were also found for mPus1p, which is directly associated with the RARE in mRAR{gamma}-target gene mRARß in vivo (19). To test whether mPus3p can also bind to this region, we performed our previously established chromatin immunoprecipitation (ChIP) assay for promoter binding by mPus1p (19). S91 cells were transfected with His-tagged mPus3p expression vector and grown in the absence or presence of CD666. Cross-linked chromatin was immunoprecipitated with anti-His or IgG control antibodies from cell lysates. As shown in Fig. 4BGo, mPus3p occupies the mRARß2 promoter site surrounding the RARE independent of ligand (normalized to input) in a similar manner as mPus1p, mRAR{gamma}, and mSRA (19).


Figure 4
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  		Fig. 4. mPus3p and mSRA Cooperatively Enhance Liganded mRAR{gamma}-Dependent Transactivation, although mPus3p Occupies the mRARß2 Promoter Site in a Ligand-Independent Manner

A, S91 cells were transfected with RARE3Luc and mSRA and mPus1p expression vectors in the absence or presence of CD666, as indicated. Luc assays were performed with the cell extracts and, after normalization, expressed as arbitrary units. *, P ≤ 0.008; **, P ≤ 0.014 (by Student’s t test analysis). B, ChIP assay showing specific, CD666-independent binding of transfected, His-tagged mPus3p with the mRARß2 promoter. S91 cells were treated for 24 h with DMSO (control) or 1 µM CD666, and subjected to cross-linking by 1% formaldehyde treatment. Chromatin was immunoprecipitated by anti-His or control IgG antibodies as indicated. mRARß2 promoter fragment was then detected by PCR analysis using the opposing primers in the schematic diagram. ß-Actin coding region serves as negative control.

 
Distinct Cellular Localization of mPus3p and hSRA
To further corroborate our interpretations, we determined the cellular localization of mPus3p, which we expected to be both nuclear, consistent with its coactivator activity, and cytoplasmic, because mPus3p is likely active in mitochondria (40). In the absence of any available Pus3p antibodies, cells were transfected with mPus3p-enhanced GFP (eGFP) expression vectors, and protein localization was analyzed by fluorescence microscopy (Fig. 5AGo). mPus3p was indeed cytoplasmic and also nuclear to a varying extent, but interestingly, in the nucleus it was localized in an unknown subcompartment that appears to exclude the nucleoli (19). Presence of ligand did not alter this pattern (data not shown). These results differ dramatically from mPus1p, which is primarily nuclear, and diffusely present throughout the nucleus, including the nucleoli. Importantly, however, mPus3p, and as shown earlier, mPus1p and mRAR{gamma} (19), at least all partially colocalize in the nucleus. Our results also suggest that, unlike mPus1p, mPus3p is involved in mitochondrial but not nucleolar tRNA processing (41).


Figure 5
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  		Fig. 5. Distinct Cellular Localization of mPus3p and hSRA

A, Fluorescence microscopy images of two representative transfected 3T3 cells showing that mPus3p-eGFP is primarily expressed in the cytoplasm, likely the site of mitochondria where it is known to reside, and in an unknown nuclear subcompartment that appears to exclude the nucleolus. DAPI staining shows the nuclei of all cells in the field. Magnification, x40. B, In situ hybridization with antisense-hSRA probe of representative MCF-7 cells transfected with hSRA expression vector in the presence of E2 (10 nM) shows that hSRA in the majority of cells (~90%) is predominantly cytoplasmic, that is mostly, or completely, absent from nuclei (indicated by black arrows). However, in a fraction of cells (~10%), hSRA is detectable in nuclei in a distinctly speckled pattern (indicated by white arrows; note mitotic cell in middle panel). Magnification, x40. C, Same experiment as in B with sense (se)-hSRA probe gives no detectable signal, showing that there was no hybridization with plasmid or genomic DNA. D, Same experiment as in B in the presence of 5 µM Tam (24 h) reveals a more diffuse distribution of hSRA throughout the entire cell, with some speckling still visible, although less clearly so.

 
We next focused on the cellular localization of hSRA. Although its association with NRs and its activity as NR coactivator suggests that it is a nuclear RNA, as previously reported (42), others reported a particular 5' splice variant that could generate a protein, suggestive of a cytoplasmic localization (13, 14). To address this issue, we used in situ RNA hybridization techniques to detect transfected (noncoding) hSRA in MCF-7 cells in the presence of E2 (endogenous hSRA was not detected by this technique) (data not shown). hSRA was visualized by hybridizing the slides with digoxigenin (DIG)-labeled antisense (as) hSRA RNA probe followed with anti-DIG-alkaline phosphatase antibody and addition of chromogenic substrate. As shown in Fig. 5BGo, hSRA was cytoplasmic in the majority of cells (~90%), but in the remainder it was localized in the nucleus in a distinctly speckled pattern, suggesting that hSRA is organized in yet another nuclear subcompartment. The results were specific for hSRA RNA, because control slides hybridized with a sense (se) hSRA probe gave no signal, showing that there was no hybridization with genomic or plasmid DNA (Fig. 5CGo). Reliability of our assay was confirmed in another control experiment in which cells overexpressing a small nuclear viral RNA showed expected, uniform nuclear localization and complete absence from the cytoplasm (data not shown). In the presence of 5 µM ER{alpha} antagonist tamoxifen (Tam), speckling remained visible although somewhat less distinctly so, and hSRA became more diffusely distributed throughout the cell, including the nucleus (Fig. 5DGo). Nuclear speckles are indicative of self-organizing regions where mRNA processing and transcription complexes form (43), and thus this result may indicate assembly or recruitment of hSRA into these subcompartments. These combined results consistently support a model in which mPus1p, mPus3p, SRA, and mRAR{gamma} form a dynamic complex in the nucleus.

Identification of a Commonly Targeted Position in hSRA that Controls Extent of Modification
Until now, the presence and the location of pseudouridylated positions in SRA in vivo has remained unknown. To address this issue, RNA from MCF-7 cells was isolated and partially treated with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and modified positions in the essential hSRA core region were probed by reverse transcription (RT) primer-extension analysis. CMCT forms a covalent bond with {Psi}, and the RT reaction stops 1 nucleotide 3' of the CMCT-altered modification. Despite the high level of secondary structure of hSRA, which greatly hindered analysis (data not shown), we successfully identified U206 as the first in vivo-modified position in endogenous hSRA (Fig. 6AGo).


Figure 6
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  		Fig. 6. Identification of an In Vivo-Modified Position in hSRA that Is a Likely Target for Pus1p and Pus3p, and that, when Mutated to A, Causes Hyperpseudouridylation and a Switch that Changes hSRA from Coactivator to a Molecule with Dominant-Negative Activity

A, Primer extension analysis of CMCT and control-treated endogenous hSRA in MCF-7 cells shows that U206 in the hSRA core sequence is pseudouridylated in vivo, as indicated by the specific stop signal that is generated in the CMCT-treated just before the modified U. On the right side is a control hSRA sequencing reaction that serves as position reference. B, Time-dependent in vitro pseudouridylation assay of hSRA-U206A with mPus1p (top) and mPus3p (bottom) shows that this substrate is more extensively modified than hSRA by both enzymes to a total of 10 and about eight positions after 180 min, respectively, despite removal of a modified site by mutagenesis. Additionally added enzyme after 90 min does not significantly increase number of modified positions. When hSRA-U206A is sequentially modified by mPus1p (90 min) and then by mPus3p, saturation of modification is reached after 270 min with 16 modified positions (top). This process is again asymmetrical because when hSRA is first modified by mPus3p (90 min) and then by mPus1p, no additional modification takes place (bottom). Background activity of similar reactions performed with control ß-galactosidase was subtracted from all time points (data not shown). C, LNCaP cells were transfected with MMTVLuc and hSRA, hSRA-U206A, or control expression vectors and DHT (50 nM) as indicated. Luc assays were performed with the cell extracts and, after normalization, expressed as arbitrary Luc units. Liganded hAR induces Luc activity in the presence of DHT, which, as expected, is robustly enhanced by cotransfected hSRA vector. In contrast, cotransfected hSRA-U206A strongly repressed induction (left), whereas neither hSRA or hSRA-U206A had any significant effect on SRA-independent E2FLuc (right). *, P ≤ 0.02; **, P ≤ 0.001 (by Student’s t test analysis). D, Similar experiment as in C except that MCF-7 cells were used with ERELuc in the presence or absence of E2 (10 nM). Here too, ligand-dependent hER{alpha}-transactivation of ERELuc is strongly enhanced by cotransfected hSRA and repressed by cotransfected hSRA-U206A vectors (left), whereas again no significant effects were seen on activity of E2FLuc (right). * and **, P ≤ 0.001 (by Student’s t test analysis). E, RT-PCR analysis showing that hER{alpha}-dependent induction of endogenous c-Myc via treatment with E2 (24 h; 10 nM) in MCF-7 cells is increased by transfected hSRA and reduced by transfected hSRA-U206A relative to transfected control expression vectors. ß-Actin serves as RNA control.

 
Next, we wanted to verify that this position is a target for mPus3p and/or mPus1p. Site-directed mutagenesis was used to change the uridine at 206 to an adenosine to create mutant hSRA-U206A. We expected that if U206 was also modified by these enzymes in vitro, hSRA-U206A would generate one fewer {Psi} than hSRA in our standard pseudouridylation assay. In contrast, however, hSRA-U206A became hyperpseudouridylated with the total number of {Psi}s generated by mPus1p and mPus3p reaching 10 and about eight {Psi}s, respectively (Fig. 6BGo). The amount of enzyme was again not rate-limiting, because addition of more of the same PUS after 90 min had no, or only very little, effect. We also observed again that premodification by mPus1p allowed further modification by mPus3p to 16 {Psi}s, whereas premodification by mPus3p almost completely prevented further modification by mPus1p, to only eight {Psi}s. These results suggest that U206 is a common target for mPus1p and mPus3p and that, after it is modified, it controls the extent of modification.

Mutation of U206 to A Switches hSRA from Coactivator to a Molecule with Dominant-Negative Activity
To test whether hyperpseudouridylation of U206A-hSRA affects its function as coactivator, we cotransfected expression vectors for hSRA, hSRA-U206A, or vector control in LNCaP and MCF-7 cells with appropriate reporter plasmids to determine activation of liganded endogenous hAR and hER{alpha}, respectively. These receptors were chosen, because whereas Pus3p would not be relevant here, both Pus1p and hSRA strongly increase transactivation by these endogenous NRs in these cells (Refs. 12 and 19 and data not shown). As shown in Fig. 6CGo, dihydrotestosterone (DHT) induced hAR-mediated induction of the MMTVLuc reporter gene, and as expected, cotransfected hSRA vector significantly enhanced this effect. However, cotransfection with hSRA-U206A vector strongly repressed AR-dependent transactivation, showing that hSRA-U206A has dominant-negative activity. A similar result was obtained for hER{alpha}-dependent activity (Fig. 6DGo). In the presence of E2, reporter gene activity was induced by activated ER{alpha}, which was further increased by cotransfected hSRA expression vector, but cotransfected hSRA-U206A again strongly repressed induction of Luc activity. No significant effects of either transfected hSRA or hSRA-U206A vectors were found on the activity of NR and SRA-independent E2F2Luc reporter plasmid (12), showing that both the activator and dominant-negative activity of hSRA and hSRA-U206A, respectively, were specific (Fig. 6Go, C and D). In addition, hSRA-U206A has a similar cellular localization pattern and expression level as hSRA (data not shown).

Next, this effect was confirmed for expression of endogenous ER{alpha}-target gene c-myc (44, 45). MCF-7 cells were transfected as above, RNA was isolated, and c-Myc levels were determined by RT-PCR analysis. As shown in Fig. 6EGo, c-Myc is induced by E2 in vector control-transfected cells, and levels are further increased when hSRA expression vector is cotransfected. However, c-Myc levels are again suppressed when cotransfected with hSRA-U206A expression vector. These results establish hSRA-U206A as a molecule with dominant-negative activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previously, it was established that mPus1p-dependent pseudouridylation of SRA is a novel type of posttranscriptional modification of a coactivator complex that is important for NR activity (19). These findings generated many new questions with regard to potential involvement of other PUSs on modification of SRA and the contributions of {Psi}s to SRA function. We showed that mPus3p is a novel coactivator with comparable liganded NR-enhancing activities as mPus1p, except that in contrast to mPus1p, mPus3p had no effect on sex steroid receptors. The latter is likely due to the inability of mPus3p to bind with these receptors, which is essential for mPus1p coactivator activity (19). Sex steroid receptors contain specific sequences not found in other NRs that play a role in transactivation, and it is possible that these directly or indirectly interfere with binding to the DBD (46). Our findings suggest that mPus3p, as shown for mPus1p (37), plays an as-yet-unrecognized important physiological role. Deficient Pus3p activity in yeast cells causes a slow growth phenotype (40) and decreases in in vivo recoding efficiency (47), and it was also implicated in a particular form of mitotic recombination of ribosomal DNA repeats (48), but unfortunately little is known about its role in higher organisms. The coactivator activities of mPus3p and mPus1p are not likely redundant, because depletion of either factor resulted in reduced mRAR{gamma}-dependent induction of target gene mRARß (Ref. 19 and the present study). Future gene knockout studies in mammalian organisms will be required to better assess the physiological relevance of Pus3p.

Based on their different tRNA position specificities, mPus3p was expected to modify SRA in different positions than mPus1p (40, 49). To address this issue, we performed pseudouridylation assays with SRA substrates and mPus1p and mPus3p, and consistent with their known behavior on tRNA, we found that they recognize different sites, although some were also commonly targeted. This may be due to the much larger size of SRA, which can likely fold into several domains that resemble tRNA-like stem-loop structures, some of which may be recognized by both enzymes (Fig. 7Go). However, our evidence also shows that sequential modification of SRA by these enzymes is an asymmetrical process, suggesting that mPus3p-introduced pseudouridylation alters the structure of SRA in such a way that it is no longer recognized by mPus1p. These findings may provide new insight into the mechanism of SRA-enhanced NR transactivation. SRA is associated with multiple proteins that can both result in activation or repression of NR signaling (12, 20, 21, 22), and it is conceivable that pseudouridylation of SRA alters its affinity for these, and/or other as-yet-uncharacterized SRA-binding proteins (22) with significant consequences for NR activity. Our findings suggest a highly complex relationship between mPus1p, mPus3p, and SRA, and the extent and type of modification where binding of coactivators and corepressors to SRA will be dependent on the relative expression and order of engagement of these enzymes with their substrate. In addition, cellular localization of mPus1p, mPus3p, and hSRA is likely another contributing factor to NR signaling. It is striking that, in contrast to mPus1p, mPus3p is readily detectable in the cytoplasm, almost certainly the mitochondria where it is known to modify tRNAs (40), whereas a varying amount is localized in an unknown nuclear subcompartment where it functions as coactivator. SRA was thought to be a nuclear RNA based on its direct association with nuclear receptors. However, our results show that hSRA is mainly cytoplasmic where it may give rise to a translated protein with unknown function in case of certain splice variants (13, 14), and only nuclear in a relatively small proportion of cells where it functions as NR coactivator. Although it is possible that endogenous hSRA is more nuclear than transfected hSRA, the pattern observed in our experiments is suggestive of trafficking of SRA and mPus3p between the cytoplasm and the nucleus where they perform different tasks, and in that regard, SRA-binding protein SLIRP is also thought to shuttle between the mitochondria and the nucleus (22).


Figure 7
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  		Fig. 7. Structure-Function Model of hSRA Core Sequence {Psi}206 Is Located in STR5, a Highly Stable Hairpin Structure Predicted to Form in the Most Stable Conformation of the hSRA Core Sequence, As Shown Here (mfold, version 3.2; Ref. 60 )

The energy and secondary structure differences between STR5 in hSRA and hSRA-U206A are relatively minor, and do not likely explain the observed large biochemical and functional effects. Instead, it is proposed that {Psi}206 stabilizes stems I and II in a higher order conformation through the specific stacking-enhancing qualities of {Psi}, which may not occur if U206 was not pseudouridylated or mutated to A. Specific spatial constraints of stems I and II may have to be met for appropriate recognition by Pus1p and Pus3p, which in turn determines whether hSRA functions as coactivator or corepressor. Location of previously characterized STR7 (22 ) is also indicated.

 
Inside the nucleus, hSRA is organized in a speckled pattern, the probable result of a self-organizing process (43). Nuclear speckles are typically divided into three main types: 1) interchromatin granules (IGs) that contain high levels of small nuclear ribonucleoproteins and non-small nuclear ribonucleoprotein splicing factors; 2) perichromatin fibrils that contain less splice factors and appear more diffusely nucleoplasmic; and 3) coiled bodies, which are similar to IGs but do not contain splice factor SC35. The role of these nuclear subcompartments is not fully understood, but several studies have suggested that the 20–40 nuclear speckles not only contain pre-mRNA splicing components but that they are peripheral to sites of active splicing and RNA polymerase II-dependent transcription (50, 51, 52, 53). Some of these structures are quite stable, consistent with our observations that Tam did not completely dissolve hSRA speckling, although the more diffuse distribution of hSRA throughout the cell in the presence of Tam indicates that activated hER{alpha} may be involved in the cellular organization of hSRA. AR, ER, GR, SRC-1, TIF-2, and CBP are also organized in nuclear speckles that number 250–400 (54, 55). ANT-1, a coactivator of GR and AR AF-1 activity is, similar to what we found for hSRA, organized in 15–20 speckles that are spatially distinct from, but surrounded by AR speckles (54). Based on this model, liganded ER{alpha} and coactivators like SRC-1 may form in the NR compartment, which then contacts the peripheral zone of the IGs that contain additional coactivators like SRA. After this occurs, transcription/splicing takes place (54). It will be worthwhile pursuing hypothesized colocalization of hSRA with these splicing and transcription factors, because this is an important step in productive NR signaling.

We also for the first time identified an in vivo pseudouridylated position in endogenous hSRA at position U206 in the critical core region. U206 is located in STR5 (Fig. 7Go), one of several predicted stem-loop structures (15). When we mutagenized U206 to A, hSRA-U206A became hyperpseudouridylated by both mPus1p and mPus3p. These results not only strongly suggest that U206 is one of few commonly targeted positions that we predicted to exist earlier, but also that it plays a key role in controlling ordered modification of SRA. As a functional consequence, we showed that U206A-hSRA had lost its coactivator activity and instead had acquired specific dominant-negative properties. The position of {Psi}206 in STR5 in the predicted structure model, and the minor differences in stability between STR5 in hSRA and hSRA-U206A relative to complete core sequence (Fig. 7Go), make it extremely unlikely that the single base substitution in U206A-hSRA caused a significant change in the secondary structure of hSRA as an explanation for the dramatic change in function of hSRA-U206A. Indeed, an earlier report showed that functionally, much larger changes in SRA are tolerated (15). A more feasible mechanism is that {Psi}206 aids in the higher order conformation of stems I and II in STR5 (Fig. 7Go). {Psi}s are known to enhance base-stacking (28) and make single-stranded RNA more rigid (56). Also, noncanonical A-C base pairs have been found directly adjacent to A-{Psi} base pairs in tRNA to help stabilize a stem-loop structure (31), which may also occur in STR5 (Fig. 7Go). We propose that if U206 is not pseudouridylated or mutated to A, stems I and II have more rotational freedom, resulting in unmasking of new positions and hyperpseudouridylation by mPus1p and mPus3p. This in turn may interfere with binding to other SRA-binding proteins that control its function as a scaffold for repressors or activators. Thus, {Psi}206 can be regarded as a posttranscriptional activator-repressor switch that constitutes a novel mechanism of regulation of hSRA activity and subsequent NR signaling. STR5 appears to be the second identified important local structure element besides SLIRP-binding STR7 (Fig. 7Go) that determines the functionality of hSRA, showing the intimate relationship between pseudouridylation and higher order structures in hSRA, and its role in activation and repression of NR activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture, Transfection, and Luc Assay
S91, 3T3, MCF-7, and LNCaP cells were obtained from the American Type Culture Collection (Manassas, VA) and grown per the supplier’s recommendations, or as previously described (19).

Transient transfection assays were carried out in six-well plates (Costar) at around 80% confluence using Lipofectamine Plus reagent according to the manufacturer’s protocol (Invitrogen, San Diego, CA) with minor modifications. Briefly, plasmids were diluted into 100 µl serum-free DMEM and mixed with 3 µl PLUS reagent. After 15 min, 3 µl LipofectAMINE reagent, which was added to another 100 µl serum-free DMEM, was mixed with the diluted plasmids. After 45 min, 1 ml of serum-free DMEM was added and then carefully placed into a well that was freshly aspirated. After 6 h, the transfection medium was removed and the cells were incubated with charcoal-stripped complete growth medium. The next day, cells were treated with 0.1% dimethylsulfoxide (DMSO), or ligands [10 nM E2, 50 nM DHT, progesterone, 1 µM thyroid hormone, dexamethasone, 0.1 µM 1{alpha},25-dihydroxyvitamin D3 (Sigma-Aldrich, St. Louis, MO); 1 µM CD666 (Galderma R&D, Sophia Antipolis, France)], and after 24 h, the cells were harvested, and Luc activity was determined after normalization for protein concentration in the cell lysate, as described previously (19). For ER transfections, cells were maintained in phenol red-free DMEM. Results show the mean ± SD of representative experiments done in triplicate and repeated at least three times. A total of 0.2–0.4 µg plasmid/well was transfected, and in all cases the total amounts of DNA were kept constant through supplementing with empty vector. The concentration of siRNA-mPus3p (ID 182550; Ambion, Austin, TX) and siRNA-control (silencer control 1; Ambion) was 60 nM and was added to the plasmid mix where needed.

Construction of Plasmid Vectors and Site-Directed Mutagenesis
All reporter plasmids and mPus1p, noncoding SRA, and NR-vectors used for expression in bacterial or mammalian cells have been described previously (12, 19, 20). Note that the hSRA cDNA used does not generate protein and only functions as RNA (12, 15). mPus3p cDNA (27) was cloned into the same expression vector (pcDNA3.1) as for mPus1p using standard techniques. hSRA-U206A was generated by using the GeneEditor mutagenesis system according to the manufacturer’s instructions (Promega, Madison, WI). Primer sequences will be provided on request. For transfection analysis, the NheI-KpnI fragment of hSRA and hSRA-U206A was subcloned into pcDNA3.1 (Invitrogen).

RNA Expression Analysis
RT-PCR analysis to detect endogenous ß-actin and mRARß in S91 cells has been described previously (19). To detect c-Myc in MCF-7 cells, a similar protocol was followed except that cells were treated with 10 nM E2. c-Myc PCR product (756 bp) was detected by standard agarose gel electrophoresis. Primer sequences were as follows: c-Myc, forward, 5'-ATGCCCCTCAACGTTAGCTTCACC; reverse, 5'-AGAGTCGCTGCTGGTGGTGGG.

Coimmunoprecipitation and Protein-Protein Binding Assay
MCF-7 and S91 cells were transfected in 15-cm plates with His-tagged pcDNA3.1-mPus3p expression vector. After 24-h treatment with 50 nM E2 or 1 µM CD666, respectively, cells were lysed in cell lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 50 mM NaF, 2 µg/ml aprotinin/leupeptin, 0.2 mM phenylmethylsulfonylfluoride (PMSF), 1 mM NaVO4]. Immunoprecipitation was performed with anti-His antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and detection of coimmunoprecipitated hER{alpha} or mRAR{gamma}, respectively, was detected by Western blot as described previously (19).

Generation of GST and GST-tagged mRAR{gamma}-DBD and mRAR{gamma}-LBD proteins, and preparation of affinity columns have been described previously (19). mPus3p was generated in T7 TnT reticulocyte lysate with [35S]Met (1175 Ci/mmol) following the manufacturer’s instructions (Promega). Binding of [35S]mPus3p to the columns and detection of bound material was done as described previously (19).

ChIP Assays
ChIP assays were performed as described previously (19) with only minor modifications. Briefly, S91 cells were transfected with His-tagged mPus3p expression vector followed by 24-h treatment with 0.1% DMSO or 1 µM CD666, and then cross-linked with 1% formaldehyde. After washing, cell pellets were sonicated. Supernatants were used as inputs and the remainder diluted 3-fold in immunoprecipitation buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1x protease inhibitor mixture]. This diluted fraction was precleared with 20 µg sheared salmon sperm DNA, 30 µl mouse IgG (Santa Cruz Biotechnology), and 50 µl of 50% protein A-agarose beads (Santa Cruz Biotechnology). Immunoprecipitation was performed overnight at 4 C with 20 µl anti-His antibody (Santa Cruz Biotechnology). Complexes were recovered by 2-h incubation at 4 C with 5 µg sheared salmon sperm DNA and 50 µl protein A-agarose beads. Precipitation of chromatin complexes and reversal of formaldehyde cross-linking, purification of DNA fragments, and amplification of ß-actin coding region and mRARß2 promoter by PCR was done as described previously (19).

Preparation of Nuclear Extracts
Nuclear extracts from Epstein-Barr virus-immortalized lymphoblastoid cells derived from a MLASA patient and normal sibling control (39) were prepared as described previously with minor modifications (57). Briefly, 50 µl cell pellet was suspended in 150 µl of 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT) at 4 C for 5 min. Next, cells were lysed by passing through a 25-gauge needle and spun at 12,000 x g, and pelleted nuclei were suspended in 250 µl of 20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, and 25% glycerol, and incubated at 4 C for 30 min. The mixture was then centrifuged at 12,000 x g for 5 min, and the resulting supernatant was dialyzed against 20 mM HEPES (pH 7.9), 0.1 M KCl, 0.2 EDTA, 0.5 mM PMSF, 0.5 mM DTT, and 20% glycerol, for 18 h at 4 C. After determining protein concentrations (1.1–1.4 µg/µl), nuclear extracts were kept frozen at –80 C until used.

Pseudouridylation Assays
Plasmid DNAs containing the SRA templates were digested with EcoRV (hSRA, hSRA-U206A) or NotI (mSRA). Templates were transcribed in the presence of 5-[3H]UTP with T7 RNA polymerase (Promega), and the RNA was isolated. The pseudouridylation reactions were carried out as described previously (27) for the times noted except that the RNA substrates, without enzyme, extract, or DTT, were heated to 78 C for 2 min and allowed to cool slowly to 37 C. Either nuclear extracts, purified recombinant mPus1p [2.1 µg (19)], or recombinant mPus3p (1.8–2.0 µg) generated in vitro using T7 TNT rabbit reticulocyte lysate and purified with MagZ beads (Promega), were incubated with the substrates. The tritium-release assay has been described previously (19). Triplicates were done on each sample, and background counts were subtracted.

To map in vivo-modified sites in hSRA, a previously described method was used (58) with modifications. Briefly, 25 µg total RNA from MCF-7 cells was dissolved in 20 µl water and mixed with 80 µl BEU buffer [7 M urea, 4 mM EDTA, 50 mM bicine (pH 8.5)] and 20 µl of 1 M CMCT (Sigma-Aldrich) at 37 C for 20 min. In a control reaction, CMCT was replaced by same volume of BEU buffer. Next, RNA was precipitated by adding 2 µl pellet paint coprecipitant (Novagen, Madison, WI), 50 µl of 3 M NaAc, and 600 µl ethanol, and dried pellets were dissolved in 50 µl SC solution [50 mM Na2CO3 (pH 10.4), 2 mM EDTA] at 37 C for 4 h. RNA was then again precipitated and dissolved into 20 µl H2O. Primer extension assay (primer sequence, 5'-ACTGACCTCAGTCACATGGTC), and standard dideoxy sequence reactions on hSRA plasmid DNA that serve as position markers, were performed as described previously (58).

Fluorescence Microscopy
3T3 cells were seeded on a two-well glass chamber slide. pN3-mPus3p-eGFP vector was transfected using Effectine Transfection reagent according to the manufacturer’s protocol (Qiagen, Valencia, CA). After 24 h, cells were analyzed by fluorescence microscopy. eGFP was detected via an fluorescein isothiocyanate filter and 4',6'-diamidino-2-phenylindole (DAPI) was analyzed via a DAPI filter.

In Situ Hybridization Assay
Localization of hSRA mRNA was determined as described by Basyuk et al. (59) with modifications. Briefly, MCF-7 cells grown on chamber slides were transfected with hSRA by Effectene transfection reagent (Qiagen). After 24 h, cells were fixed for 1 h in 4% paraformaldehyde in PBS (pH 9), and then washed in PBS (pH 7.0). Next, cells were permeabilized in PBS/1% Triton X-100 for 20 min, washed, and soaked for 15 min in 50% formamide containing 5x standard saline citrate (SSC). Then, 100 µl hybridization buffer (50% deionized formamide, 10% dextran sulfate, 5x SSC, 250 µg/ml yeast tRNA, and 0.02% RNase-free BSA) containing 200–400 ng of appropriate DIG-labeled probes were added to slide sections and incubated in a humid chamber (5x SSC, 50% formamide) overnight at 55 C. Slides were dipped in 2x SSC and 50% formamide at 55 C for 15 min, and then washed for another 30 min under the same conditions, and then washed in 0.2x SSC and 50% formamide at 55 C followed by 5-min washing in only 0.2x SSC at room temperature. After 1-h incubation in a blocking solution [100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% sheep serum], slides were incubated in the same solution but with 1% normal sheep serum and 1:100 dilution of anti-DIG-alkaline phosphatase-conjugated antibody (Roche, Basel, Switzerland) overnight at 4 C. Slides were washed in same buffer and treated with 200 µl nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate developing reagent (KPL, Gaithersburg, MD) for 2–4 h in darkness. Slides were briefly washed in TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA], rinsed with water, air dried, and mounted on glass slides with few drops of 50% glycerol and sealed with clear nail polish. Slides were viewed by light microscopy and images were stored as JPG files. To prepare hSRA-specific DIG-labeled probe, a 300-bp fragment was PCR amplified using the following primers that contain T7 RNA polymerase binding site sequences at the 5' end of the 5' primer): for antisense template, T7AS-F, 5'-TAATACGACTCACTATAGGGCTGAAAACAGACTCCTCTTTTCTGC-3', and T7AS-R, 5'-GCCACACAAGGAAGCAGGTATGTGATG-3'; sensetemplate, T7SS-F, 5'-TAATACGACTCACTATAGGGGCCACACAAGGAAGCAGGTATG-3', and T7SS-R, CTGAAAACAGACTCCTCTTTTCTGC-3'. RNA transcription was carried out on the PCR template using Digoxigenin RNA labeling kit (Roche) using the manufacturer’s protocol. Template DNA was removed by RNase-free DNase treatment, and RNA was ethanol precipitated and resuspended in diethylpyrocarbonate-treated water. Probes were heated at 80 C for 1 min before adding in to the hybridization mixture.


   ACKNOWLEDGMENTS
 
We thank Rainer B. Lanz and Bert W. O’Malley (Baylor College of Medicine, Houston, TX) for providing us with original hSRA, mSRA, and PRß expression vectors and ERE-Luc reporter plasmid, and Gerald Denis and Tai Chen (Boston University School of Medicine, Boston, MA) for E2F-Luc and VDRE4-Luc reporter plasmids, respectively. We also thank Galderma R&D (Sophia Antipolis, France) for providing us with CD666.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant RO1 CA76406 (to R.A.S.), NIH Grant RO1 DK074368 (to N.F.-G.), and a Research and Productive Scholarship Grant from the University of South Carolina (to J.R.P.).

The authors have nothing to disclose.

First Published Online December 14, 2006

Abbreviations: AR, Androgen receptor; ChIP, chromatin immunoprecipitation; CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate; DAPI, 4',6'-diamidino-2-phenylindole; DBD, DNA-binding domain; DHT, dihydrotestosterone; DIG, digoxigenin; DMSO, dimethylsulfoxide; DTT, dithiothreitol; E2, 17ß-estradiol; eGFP, enhanced green fluorescent protein; ER, estrogen receptor; GR, glucocorticoid receptor; GST, glutathione S-transferase; h, human; IG, interchromatin granule; LBD, ligand-binding do-main; Luc, Luciferase; m, mouse; MLASA, mitochondrial myopathy and sideroblastic anemia; NR, nuclear receptor; PMSF, phenylmethylsulfonylfluoride; PR, progesterone re- ceptor; {Psi}, pseudouridine; PUS, pseudouridine synthase; RAR, retinoic acid receptor; RARE, retinoic acid response element; RT, reverse transcription; siRNA, short interfering RNA; SRA, steroid receptor RNA activator; SSC, standard saline citrate; STR, stem-loop structure; Tam, tamoxifen; TR, thyroid hormone receptor; tRNA, transfer RNA; VDR, vitamin D3 receptor.

Received for publication October 2, 2006. Accepted for publication December 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  RARβ  |  RARγ  |  VDR  |  ERα  |  GR  |  PR  |  AR
Coregulators:   PUS1  |  SRA
Ligands:   17β-Estradiol  |  Dihydrotestosterone



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