1. Volume 15, Issue 4, Pages 549-558 (August 2004)
Regulation of Nuclear Receptor Activity by a Pseudouridine Synthase through Posttranscriptional Modification of Steroid Receptor RNA Activator 
Xiansi Zhao, Jeffrey R. Patton, Shannon L. Davis, Brian Florence, Sarah J. Ames, Remco A. Spanjaard 
Molecular Cell 
Volume 15, Issue 4, Pages (August 2004)
DOI: /j.molcel

2. Figure 1
mPus1p Is a Specific Coactivator of mRARγ and Other NR-Dependent Transactivation
(A) Ligand- and dose-dependent effect of mPus1p-enhanced mRARγ-mediated transcriptional activation. S91 cells were transfected with RARE3Luc and different doses of mPus1p plasmids and ligand (1 μM CD666) as indicated. Luciferase (Luc) assays were performed with the cell extracts, and after normalization, expressed as arbitrary Luc units. *p ≤ 0.044; **p ≤ (by Student's t test analysis).
(B) Antisense mPus1p (asODN-P), but not scrambled control (scODN-P) oligodeoxynucleotides, suppress mRARγ-dependent transactivation. Cells were transfected with RARE3Luc and increasing doses of mPus1p plasmids, and CD666, asODN-P, or scODN-P, as indicated, and subjected to Luc assays. *p ≤ ; **p ≤ (by Student's t test analysis).
(C) Cells transfected with RARE3Luc and mPus1p vector show that CD666 and mPus1p-dependent mRARγ activity is inhibited by 10-fold excess RARγ-antagonist CD2366 (left; *p ≤ by Student's t test analysis). mPus1p does not stimulate mRARγ activity in the absence of a RARE in the target promoter of ΔRARELuc reporter (middle), nor does mPus1p nonspecifically increase RSVLuc activity (right).
(D) RT-PCR analysis showing that mRARγ-dependent induction of endogenous mRARβ via treatment with CD666 (16 hr, 1 μM) is inhibited in cells that were treated with asODN-P, but not with scODN-P, as indicated. β-actin serves as RNA control.
(E) Ligand-dependent (T3, thyroid hormone; Dex, dexamethasone; DHT, dihydrotestosterone; Prog, progesterone; E2, 17β-estradiol), mPus1p-enhanced transactivation of TRβ, GR, AR, PRβ, and ERα-transactivated Luc reporter plasmids (*p ≤ , p ≤ , p ≤ 0.015, p ≤ , and p ≤ by Student's t test analysis, left to right, respectively).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Binding Domain-Mapping Analysis of mPus1p and mRARγ
(A) Binding of [35S]mRARγ and deletion mutants to GST-mPus1p was assessed by SDS-PAGE and autoradiography. mRARγ domains A–E, including domain C (black), which contains the DBD with Zn fingers I and II, and the position of the T/A box and H12 are indicated. Binding with mPus1p is lost in mutants 1–106 and 1–89 (gray fill), where Zn finger I is disrupted. The GST-mRARγ-LBD fragment (bottom, gray fill) is unable to bind [35S]mPus1p in reciprocal experiment, confirming the previous analysis.
(B) Pull-down experiment showing that His and GST-mPus1p specifically bind mRARγ and hERα from indicated cell lysates, respectively. Immunoblot with the indicated antibodies.
(C) Binding of [35S]mPus1p region 211–394, which was originally identified by yeast two-hybrid assay (boxed fill) and other deletion mutants to GST-mRARγ-DBD. D112 is critical for Ψ formation. Binding is lost in mutants 272–394 and 282–394 (gray fill), showing that region 261–271, which includes hydrophobic sequence 263–269, is critically important.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
mPus1p Specifically Pseudouridylates SRA In Vitro, and When Coexpressed Cooperatively Enhances mRARγ-Dependent Transcriptional Activity
(A) Assay showing that after 2 hr incubation with recombinant mPus1p (700 ng), 4 positions in hSRA and mSRA and 3 positions in tRNAIle are specifically modified (χ2 p ≤ 0.078, , and , respectively). β-galactosidase (β-gal) and tRNAAla serve as negative controls; tRNAIle serves as positive control.
(B) Cotransfected mPus1p and mSRA cooperatively enhance mRARγ-dependent transactivation, but mPus1p is unable to function as coactivator in cells that have been made mSRA deficient through transfection with asODN-S but not scODN-S. S91 cells were transfected with RARE3Luc, mSRA, and mPus1p (0.1 μg) plasmid, CD666, asODN-S, or scODN-S, as indicated. Luc assays were performed with the cell extracts and after normalization, expressed as arbitrary Luc units. Left: *p ≤ ; **p ≤ ; right: *p ≤ ; **p ≤ ; ***p ≤ (by Student's t test analysis).
(C) Pull-down experiment showing that mSRA from S91 cell lysate specifically binds GST-mPus1p. RT-PCR reaction showing expected product only in +RT lane in the presence of GST-mPus1p.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
mRARγ, mPus1p, and SRA Occupy the mRARβ2 Promoter Site In a Complex In Vivo that Is Independent of Ligand
(A) ChIP assay showing specific, CD666-independent binding of mRARγ and mPus1p with the mRARβ2 promoter. Cells were treated for 24 hr with DMSO (control) or 1 μM CD666 and subjected to crosslinking by 1% formaldehyde treatment. After preclearing of lysate with IgG, chromatin was immunoprecipitated by anti-mRARγ and anti-mPus1p antibodies as indicated. mRARβ2 promoter fragment was then detected by PCR analysis using the opposing primers in the schematic diagram. Results shown are representative of three independent experiments. β-actin coding region serves as negative control. input, input DNA.
(B) Immunoprecipitation assay showing association of mSRA with mPus1p (left) and mRARγ (right). S91 cell lysates were incubated with control IgG, anti-mRARγ, or anti-mPus1p antibodies, and immunocomplexes were precipitated by binding to Protein A-agarose beads. Next, RNA was extracted and mSRA (800 bp) was detected by RT-PCR analysis. IgG and RT lanes serve as negative controls. In asODN-S treated, mSRA-deficient cells no association between mSRA and mRARγ is detected, confirming specificity of the results.
(C) Confocal microscopy image of transfected 3T3 cells showing that mPus1p-RFP is expressed in the nucleolus (no) and nucleus. mRARγ-eGFP is only present in the nucleus where it colocalizes with mPus1p-RFP. Magnification: 40×.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Generation and Validation of mPus1p Mutants with Specific Defects in mRARγ Binding (RBM5) or PUS Activity (D112A)
(A) Binding of [35S]mPus1p with point mutations in region 263–269 (substitutions are underlined relative to wild-type sequence) to GST-mRARγ-DBD. Only RBM5 is unable to bind.
(B) Pseudouridylation assay showing that mPus1p and RBM5 modify yeast tRNAIle to almost the same extent (about 2 mole Ψ/mole RNA/30 min reaction), while D112A is completely inactive. Immunoblot below, probed with anti-mPus1p antibody, shows that the lysates yielded equivalent amounts of PUSs (70 ng/reaction).
(C) Mutant D112A binds GST-mRARγ-DBD as well as mPus1p.
(D) Pull-down experiment showing that His-D112A still binds SRA as well as GST-mPus1p. Fusion proteins were incubated with mouse (mo) or human (hu) SRA, and binding was assessed by RT-PCR. Specific SRA products are indicated (please note use of different mSRA primers in top and bottom panel, generating different sized fragments).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
mRARγ Binding and PUS Activities Are Essential and Complementary Properties Required for mPus1p Coactivator Function
S91 cells were transfected with RARE3Luc, mSRA, and combinations of mPus1p, RBM5, and D112A (0.1 μg) plasmids and CD666 as indicated. Luc assays were performed with the cell extracts and after normalization, expressed as arbitrary Luc units. *p ≤ ; **p ≤ 0.030; ***p ≤ ; ****p ≤ (by Student's t test analysis).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 Model of mPus1p-SRA-NR-Dependent Signaling Axis
mPus1p serves a dual function: in the nucleolus it is involved in tRNA biogenesis and in the nucleus it acts as coactivator of NR signaling. mPus1p-dependent modification of SRA (in and/or outside the nucleolus) allows it to switch from an inactive to an active conformation. Next, the mPus1p-SRA complex binds to the DBD of the NR at the HRE of a target gene in a ligand-independent manner. When ligand is present (indicated), transcriptional activation ensues. SRA forms a potential bridge between AF-1 and SRC-1-associated AF-2 of class I NRs and possibly forms a scaffold for additional SRA binding coactivators. The specific complementary defects in mutants D112A and RBM5 are indicated.
Molecular Cell  , DOI: ( /j.molcel )