HADHB, HuR, and CP1 Bind to the Distal 3′-Untranslated Region of Human Renin mRNA and Differentially Modulate Renin Expression*

Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich “renin stability regulatory element” in the REN 3′-untranslated region (3′-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3′-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) β-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3′-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3′-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3′-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.

Renin, secreted by renal juxtaglomerular cells, is rate-limiting in angiotensin II generation and regulated by perturbations in NaCl delivery to the distal nephron and renal perfusion pressure (1). Thus, the human renin gene (REN) requires tight control to ensure expression appropriate to need. A better understanding of this is necessary in view of the importance of the renin system as a major therapeutic target in hypertension. We have found that REN expression is subject to both transcriptional (2) and post-transcriptional (3) control. A role for the 3Ј-untranslated region (3Ј-UTR) 1 of REN mRNA in cell-and tissue-specific expression has been demonstrated in transgenic studies (4). Via an action on an intermediate gene to stabilize REN mRNA, cAMP increases REN mRNA by 100-fold (3,5), reminiscent of effects on other mRNAs (6,7).
Post-transcriptional stabilization of REN mRNA could explain how, despite a weak REN promoter (28,29), REN mRNA can be detected readily in the kidney, where its source (the juxtaglomerular cells) comprises Ͻ0.01% of the renal cell population. The REN 3Ј-UTR is relatively short (196 nucleotides) and lacks classical AREs. Here we sought to identify novel REN 3Ј-UTR cis-elements and binding proteins, and found interactions involving HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) ␤-subunit), HuR, and CP1 that are likely to play a major role in the regulation of human renin expression.

EXPERIMENTAL PROCEDURES
Cell Culture-The renin-expressing human pulmonary adenocarcinoma Calu-6 cell line was maintained as described previously (30).
Transient Transfection Assays-Calu-6 cells (1.5 ϫ 10 5 ) were cotransfected with 1 g of ecdysone receptor plasmid pRXR (Invitrogen); 1 g of pLucUTR, pLucMUT, pLucSV, or pLucSV-SRE; and 500 ng of pCMV-SEAP (transfection efficiency control) using FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. After 24 h, the cells were treated with 25 M ponasterone A (Invitrogen). After a further 24 h, the cells were harvested, and luciferase was measured using the luciferase assay system from Promega. SEAP (Great Escape™ SEAP assay kit, Roche Diagnostics) was assayed in cell medium collected prior to ponasterone A addition.
RNA Electrophoretic Mobility Shift Assay (REMSA)-To generate riboprobes, pBluescript-REN-WT was linearized with HindIII for transcription with T7 RNA polymerase, and pBluescript-REN-MUT was linearized with EcoRI for transcription with T3 RNA polymerase. The pBluescript vector alone was linearized with HindIII. Linearized RNA templates were transcribed with T7 or T3 RNA polymerase (Invitrogen) in reactions containing [ 32 P]UTP (37 Ci/mmol; Amersham Biosciences), as described (39), to give transcripts with a specific activity of ϳ0.5 ϫ 10 10 cpm/g RNA. Unlabeled transcripts were synthesized as described above, but with 2.5 mM rNTPs, and quantified by polyacrylamide gel electrophoresis. Binding reactions were performed as described (41)(42)(43) with 10 g of Calu-6 cytoplasmic extract, 200 ng of GST-HADHB, 200 ng of GST-HuR, or 200 ng of cleaved CP1 and 10 5 cpm RNA (ϳ10 -20 pg). Briefly, following incubation at 22°C for 30 min, 0.3 units of RNase T1 (Roche Diagnostics) was added for 10 min, followed by 50 g of heparin (Sigma) for 10 min. Samples were electrophoresed on a 6% acrylamide gel, dried, and analyzed using a PhosphorImager (Amersham Biosciences). In RNA competition assays, a 100-fold excess of unlabeled sense RNA transcript (e.g. REN or pBluescript) was preincubated with the extract for 30 min at 22°C prior to incubation with the labeled probe as described above. In some assays, antibodies to specific RNA-binding proteins (HADHB, HuR, and CP1) were added as described (25,27) in an effort to supershift RNA-protein complexes.
UV Cross-linking of RNA-Protein Complexes-RNA-protein binding reactions were carried out as described above using 20 g of Calu-6 cytoplasmic extract or 200 ng of cleaved CP1 fusion protein and 2 ϫ 10 5 cpm RNA (15-30 pg) 32 P-labeled riboprobe (39,41,44). After adding heparin, samples were placed on ice in a microtiter tray, UV-irradiated for 30 min, incubated with 20 g of RNase A (Roche Diagnostics) at 37°C for 15 min, and boiled for 3 min in SDS sample buffer. RNAprotein complexes were separated by 10% SDS-PAGE and analyzed with a PhosphorImager using 14 C-labeled Rainbow molecular mass markers (Amersham Biosciences).
Immunoprecipitation and Reverse Transcription (RT)-PCR Assay-Calu-6 cells grown to ϳ70% confluence in 10-cm dishes were washed with PBS, trypsinized, washed again with PBS, and then lysed in 1 ml of cytoplasmic extract buffer (see REMSA procedures described above) on ice for 20 min. Lysates were centrifuged at 11,500 rpm for 10 min, and the supernatant was removed to fresh tubes. Anti-HADHB antibody (10 l), anti-HuR antibody (10 g), anti-CP1 antibody (10 g), anti-p21 WAF1 monoclonal antibody 15091A (BD Biosciences) or no antibody was added to lysates and incubated for 60 min at 4°C on a rotating wheel. Fifty l of a 50% slurry of a mixture of protein A beads (Amersham Biosciences) and protein G beads (Sigma) (pre-swollen and equilibrated in cytoplasmic extract buffer) was added to each sample and incubated for a further 60 min. After centrifugation at 4500 rpm for 2 min, the supernatants were removed; the pelleted beads were washed five times with cold cytoplasmic extract buffer; and RNA was extracted using TRIzol reagent (Invitrogen). RNA was treated with RQ1 RNasefree DNase I (Promega) to eliminate any genomic DNA, and RT was performed using random hexamers (Promega) and Superscript II (Invitrogen) following standard procedures. PCR was performed for 40 cycles, with annealing at 55°C, using primers REN-F (5Ј-gtg ggg tca tcc acc ttg ctc-3Ј) and REN-R (5Ј-cct gaa ata cat agt ccg cgc agg t-3Ј), flanking an intron site within REN cDNA to avoid any genomic DNA effect. PCR products (ϳ235 bp) were resolved on ethidium bromidestained 1.5% agarose gels.
RNA Interference (RNAi) Experiments-Small inhibitory RNA (siRNA) oligonucleotides were designed 261 and 88 nucleotides 3Ј of the first ATG codon in HADHB and HuR mRNAs, respectively. A 21-mer of each was generated with an 8-mer (cct gtc tc) tagged to the 3Ј-end for T7 priming. Selected sequences were subjected to a BLAST search to determine specificity for the HADHB and HuR genes: HADHB target mRNA, 5Ј-aa gac aug cca cau gau u-3Ј; antisense HADHB siRNA oligonucleotide template (with U residues replaced by T residues), 5Ј-aa gac ctg atg cca cat gat t cct gtc tc-3Ј; sense HADHB siRNA oligonucleotide template, 5Ј-aa aat cat gtg gca tca ggt c cct gtc tc-3Ј; HuR target mRNA, 5Ј-aa cau gac cca gga uga guu a-3Ј; antisense HuR siRNA oligonucleotide template, 5Ј-aa cat gac cca gga tga gtt a cct gtc tc; sense HuR siRNA template, 5Ј-aa taa ctc atc ctg ggt cat g cct gtc tc-3Ј; antisense nonsense siRNA oligonucleotide template, 5Ј-aa tgt cac gag att aca cca t cct gtc tc-3Ј; and sense nonsense siRNA oligonucleotide template, 5Ј-aa atg gtg taa tct cgt gac a cct gtc tc-3Ј.
siRNA transcription templates (double-stranded DNA) were generated using an Ambion siRNA kit according to the manufacturer's instructions. After annealing of T7 promoter sequence to siRNA oligonucleotides, double-stranded RNA was generated by in vitro transcription with T7 RNA polymerase. Leader sequences were removed by RNase T1 and DNase I. siRNAs were purified by phenol/chloroform extraction and quantified by absorbance at 260 nm, and integrity was determined by PAGE.
Calu-6 cells were plated at 3 ϫ 10 5 cells/well in 2-cm 6-well plates and transfected the next day with siRNA (100 nM/well) using Oligo-fectAMINE (Invitrogen) and Opti-MEM serum-free medium (Invitrogen) as recommended. After 4 h, the medium was replaced by Dulbecco's modified Eagle's medium and 5% fetal calf serum; and then 24 h post-transfection, either cells were harvested for protein, or actinomycin D (ActD) chase assay was performed, and RNA was extracted for RT-PCR.
Intracellular Localization Studies-For HuR, Calu-6 cells were cultured on Lab-Tek II chamber slides (Nalge Nunc International) and fixed with 100% ice-cold methanol. After blocking for 1 h with 5% bovine serum albumin, cells were incubated with anti-HuR antibody for 1 h and then with Alexa Fluor 594-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes, Inc.) before staining with 300 nM 4,6diamidino-2-phenylindole (Molecular Probes, Inc.) and mounted with glycerol/gelatin/PBS (Sigma). Two-channel fluorescent images were acquired on a Zeiss Axioplan 2 imaging microscope. For colocalization of HADHB and mitochondrial proteins, cells were cultured, fixed, and blocked as described above and then incubated for 1 h with antibodies to HADHB and mitochondrial proteins (1:1000; mouse MAB1273, Chemicon International, Inc.). The secondary antibodies used were Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488conjugated goat anti-mouse IgG (Molecular Probes, Inc.). Finally, the cells were stained and mounted as described above before acquiring two-and three-channel fluorescent images. CP1 localization was as described for HADHB, but with anti-CP1 antibody and mouse antibody to SR proteins (1:50; SR(1H4), Santa Cruz Biotechnology). All localizations were also tested 24 h after 10 M forskolin addition to cells.

RESULTS
The REN 3Ј-UTR Contains a cis-Element-We made a reporter construct containing the REN 3Ј-UTR immediately 3Ј of luciferase (pLucUTR) (Fig. 1A). Although the REN 3Ј-UTR does not contain classical AU-rich pentamers and nonamers or extended U-rich sequences, it is AU-rich; and we found a 34nucleotide region at the 3Ј-end with homology (Fig. 1E) to a region of sarcotoxin IIA mRNA (from Sarcophaga peregrina) that can bind a bifunctional RBP, 3-oxoacyl-CoA thiolase (15). Modeling using the mfold server 2 showed stable stem-loop formation in the REN 3Ј-UTR (Fig. 1F), with the most distal involving the REN-SRE. REN-SRE deletion altered the overall structure, but preserved the 5Ј-end stem-loop (Fig. 1F). We thus made an REN-SRE mutant (pLucMUT) lacking this AUrich region. Calu-6 cells were cotransfected with pRXR, pCMV-SEAP, and one of the luciferase constructs and then induced with ponasterone A for 24 h. The REN-SRE deletion led to a 3-fold increase in luciferase ( Fig. 2A), suggesting that it may be a destabilizing element. When placed immediately 3Ј of luciferase and 5Ј of the SV40 poly(A) sequence (pLucSV-SRE), the activity decreased by ϳ20-fold (Fig. 2B). The REN-SRE had no effect on mRNA translation in rabbit reticulocyte lysates: (1.3 Ϯ 0.2) ϫ 10 6 relative light units for wild-type REN (REN-WT) versus (0.9 Ϯ 0.1) ϫ 10 6 relative light units for mutant REN (REN-MUT) (n ϭ 3; p ϭ 0.7). Thus, the REN 3Ј-UTR can act in cis to regulate the activity of a heterologous reporter, and the REN-SRE is a destabilizer.
Yeast Three-hybrid Screen-To find REN 3Ј-UTR protein interactors, we used the yeast three-hybrid system to screen a human kidney cDNA expression library. Multiple pRH3Ј-UTR/ pYESTrp2 transformants grew on YC-Trp Ϫ /Ura Ϫ /His Ϫ plates. The pYESTrp2 library plasmid was rescued from these transformants and sequenced. One clone contained the 3Ј 30% (residues 285-410) of HADHB (GenBank TM /EBI accession number BAA22061) (Fig. 1B). Yeast cells transformed with both the pYESTrp2-HADHB (prey) and pRH3Ј-UTR (bait) plasmids ac-tivated the reporter genes HIS3, indicated by survival on minimal essential medium plates in the absence of histidine and the presence of 20 mM 3-aminotetrazolium (a histidine antagonist), and lacZ, indicated by production of a blue color in the presence of X-gal (Fig. 3A). Their growth in the presence of 5-fluoroorotic acid (data not shown) indicated that the pRH3Ј-UTR plasmid was amplified in these yeast cells, thus validating the interaction between the RNA and the pYESTrp2 library plasmid (34). In contrast, yeast cells transformed with both the pYESTrp2-HADHB and pRH3Ј (containing no insert) plasmids were unable to grow on histidine-deficient plates and could not 3Ј-UTR/Binding Proteins/Stability/Expression of Renin mRNA metabolize X-gal (Fig. 3B). The positive control was the strong interaction of the iron-responsive element protein with the iron-responsive element RNA (Fig. 3C). The REN 3Ј-UTR/ HADHB interaction was thus specific.
Interaction of Calu-6 Proteins with the REN 3Ј-UTR-Calu-6 cell cytoplasmic extracts and the REN 3Ј-UTR riboprobe (REN-WT) gave a distinct RNA-binding protein complex (RPC) in REMSA (Fig. 4A, lane 1) that could be competed with excess unlabeled REN-WT probe (lane 2), but not with excess unlabeled pBluescript (lane 3). Deletion of the REN-SRE to give the REN-MUT probe substantially reduced binding (Fig. 4A, lane 1  versus lane 6). Because binding by the latter was efficiently competed with excess unlabeled REN-WT probe (Fig. 4A, lane  7), binding motifs 5Ј of the REN-SRE may be present.
UV cross-linking revealed that multiple proteins targeted the REN 3Ј-UTR (Fig. 4B, lane 1). RPCs at ϳ47 and ϳ36 kDa corresponded to bands on Western blots with molecular masses of HADHB and HuR, respectively (Fig. 4B, *). Deletion of the REN-SRE decreased binding of the band at ϳ43 kDa and increased binding of the band at ϳ36 kDa (Fig. 4B, lane 6). The actin control confirmed equal loading on each lane (data not shown).
Binding of HADHB, HuR, and CP1 to the REN mRNA 3Ј-UTR-Recombinant GST-HADHB bound to the REN-WT 3Ј-UTR probe specifically in REMSA (Fig. 5A, lane 2), and the RPC was competed with excess (ϳ100-fold) unlabeled mRNA (REN-WT 3Ј-UTR) (lane 3), but not with this excess of pBluescript RNA (lane 4). Results for GST-HuR were similar (Fig.  5B, lanes 6 -8). Anti-HuR monoclonal antibody decreased GST-HuR binding to the REN-WT probe and gave a supershift (Fig.  5B, compare lanes 2 and 3). Anti-HuR monoclonal antibody also reduced probe binding intensity in Calu-6 extracts, with a less intense (but present) supershift (Fig. 5B, compare lanes 4  and 5). Binding of GST-HuR to REN-MUT mRNA was less than to the REN-WT probe, consistent with the involvement of the REN-SRE in binding (data not shown). These data demonstrate direct and specific binding of HADHB and HuR to the REN 3Ј-UTR.
The sequence CCCUUCCC not far from the 3Ј-end of the REN 3Ј-UTR is homologous to a consensus CP-binding sequence (CCCUCCC) (8,26,27). Because CPs are ϳ42 kDa, we tested whether the ϳ43-kDa RPC obtained upon UV crosslinking was CP1. Antibody to CP1 induced a supershift with a reduction in RPC intensity (Fig. 5C, lane 2). We made GST-CP1, cleaved it with PreScission TM protease (Amersham Biosciences), and showed in REMSA that it bound avidly to the REN-WT probe (Fig. 5C, lane 4). A supershift was produced by anti-CP1 antibody (Fig. 5C, lane 5). Cleaved CP1 bound, but less avidly, and could still be supershifted with the REN-MUT probe (Fig. 5C, lanes 7 and 8). The 3Ј-end of the C-rich motif was absent in REN-MUT, likely explaining the reduced binding (Fig. 1F). Binding was competed with excess unlabeled REN-WT RNA (Fig. 5C, lane 11), but not with vector (lane 12). Upon UV cross-linking, cleaved CP1 bound definitively to the REN-WT probe (Fig. 5D, lane 1), but less so when the REN-SRE was absent (lane 2). Immunoblotting revealed cleaved CP1 protein at ϳ43 kDa (Fig. 5D, lane 4). These diverse data strongly support CP1 being a major component of the ϳ43-kDa RPC in Fig. 4B. Analysis of HADHB, HuR, and CP1 levels showed that each increased in response to forskolin (Fig. 5E).
Effect on Renin of RNAi Directed at HuR and HADHB mRNAs-Transfection of specific siRNAs in Calu-6 cells led to a substantial down-regulation of both HADHB and HuR, with no effect on actin (Fig. 7A, upper and lower panels, respectively,  lane 2). Transfection with nonsense siRNA had no effect on  (Fig. 1A) was cotransfected with pCMV-SEAP (internal control) and the pRXR plasmid expressing the ecdysone receptor. SEAP (used to correct for transfection efficiency) was measured 24 h post-transfection when the inducer (ecdysone) was added (0 h); and 24 h later, luciferase was measured. B, shown are the results for the pLucSV-SRE construct (SV) (Fig. 1A).

FIG. 3. Binding of HADHB to the REN 3-UTR in yeast threehybrid assay.
A, yeast cells containing the pRH3Ј-UTR plasmid and a length of HADHB cDNA (pYESTrp2-HADHB) were able to grow on Trp Ϫ /Ura Ϫ /His Ϫ selective medium and to express ␤-galactosidase (blue). B, yeast cells containing pRH3Ј (no insert) and pYESTrp2-HADHB were unable to grow and did not express ␤-galactosidase. C, the iron-responsive element RNA (IRE; pRH3Ј-IRE) and the iron-responsive element protein (IRP; pYESTrp2-IRP) interacted strongly, as indicated by yeast growth on Trp Ϫ /Ura Ϫ /His Ϫ plates and ␤-galactosidase expression (blue). HADHB, HuR, or actin levels (Fig. 7A, lane 3). For HADHB siRNA, renin protein increased (Fig. 7A, upper panel, center  row, lane 2), whereas for HuR siRNA, a decrease in renin was seen (lower panel, center row, lane 2). This provides functional data consistent with the notion that HADHB and HuR modulate REN mRNA stability, resulting in changes in renin protein levels.
To evaluate the effect of HADHB and HuR on REN mRNA stability, we performed ActD chase experiments on Calu-6 cells transfected with HADHB or HuR siRNA. In control cells (no siRNA transfected), the half-life of REN mRNA was ϳ4.5 h (Fig. 7B). However, in the presence of HADHB siRNA, the REN mRNA half-life was increased to Ͼ12 h (Fig. 7B), consistent with the increase in total renin protein (Fig. 7A) and the theory that HADHB destabilizes the REN mRNA. In contrast, with HuR siRNA, the REN mRNA half-life was reduced to ϳ2.5 h (Fig. 7B), consistent with the renin protein levels (Fig. 7A) and the notion that HuR stabilizes REN mRNA.
Effect of Thiolase Inhibition on Renin Protein Accumulation-To extend our finding that 4-BCA reduced the binding of proteins to REN mRNA (Fig. 4B), we examined the effect of 4-BCA on renin protein levels. We found that addition of 4-BCA to Calu-6 cells resulted in significantly increased renin protein accumulation over the course of several hours (Fig. 8A).
Localization of HADHB, HuR, and CP1-Three-dimensional imaging microscopy showed colocalization of HADHB with mitochondrial proteins (Fig. 9A). Some HADHB was in the cytoplasm and in the nucleus within nucleoli. In contrast, HuR was widely expressed in the nucleus, but not in nucleoli, and there was little in the cytoplasm (Fig. 9B). CP1 was also in the nucleus and partly colocalized with SR proteins in speckles (Fig. 9C), which are subregions where RNA-processing factors are stored or assembled (46,47). Nonspecific staining, assessed by incubating cells with rabbit or mouse IgG instead of primary antibody as well as by incubating with no primary antibody, was negligible (data not shown). Forskolin treatment did not change the localization patterns seen (data not shown).
Localization of HADHB within the Kidney-Immunohistochemistry of mouse kidney sections with anti-HADHB antibody demonstrated particularly strong staining in the afferent arteriole proximal to the glomerulus (Fig. 10), i.e. in the juxtaglomerular cells, the major physiological source of renin. As expected, HuR, which is expressed ubiquitously, was distributed evenly across all cell types in the kidney (data not shown).

DISCUSSION
Although REN mRNA is regulated primarily at the level of mRNA turnover, the molecular mechanisms involved have remained elusive. Here we demonstrated that the REN 3Ј-UTR can regulate a heterologous reporter and that a novel AU-rich cis-element of 34 nucleotides (REN-SRE) can convey its regulatory effect to the SV40 poly(A) sequence. We then identified and characterized a novel set of REN mRNA-binding proteins: HADHB, HuR, and CP1. The binding of these to REN mRNA was regulated by forskolin, which increased whole cell levels of each. We also showed that HuR, HADHB, and CP1 each associate with REN mRNA in vivo. Each of these proteins is differentially distributed in Calu-6 cells: HADHB mainly in mitochondria; HuR in the nucleus; and CP1 broadly throughout the cell, but mostly in the nucleus. Moreover, our RNAi experiments provide functional data that support the notion that HADHB destabilizes REN mRNA, whereas HuR acts to increase REN mRNA stability.
However, several other metabolic enzymes are known to bind RNA, including glutamate dehydrogenase, NAD ϩ -dependent isocitrate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, thymidylate synthase, dihydrofolate reductase, catalase, enoyl-CoA hydratase, and thiolase (8). A number of these enzymes are NAD ϩ /NADH coenzyme-dependent; and interestingly, the NAD ϩ -binding fold of glyceraldehyde-3-phosphate dehydrogenase, which comprises a ␤␣␤ structure (Rossmann fold), is sufficient to confer RNA binding to AU-rich sequences (50). We found that thiolase activity may be important in renin production because the thiolase inhibitor 4-BCA increased renin progressively in cultured Calu-6 cells. Also of interest, a weak 4-BCA-sensitive band at ϳ39 kDa obtained upon UV cross-linking is the size of human 3-oxoacyl-CoA thiolase. In S. peregrina, 3-oxoacyl-CoA thiolase binds to sarcotoxin IIA mRNA in a region (15) that has high homology to the distal end of the REN 3Ј-UTR. Its effect on mRNA stability in S. peregrina has not, however, been tested. Consequently, further identification and characterization of the proteins binding at ϳ29 and  REN-SRE deleted; lanes 6 -8), and REMSA was performed, followed by analysis using a PhosphorImager (see "Experimental Procedures"). In specific lanes, an ϳ100-fold excess of unlabeled (cold) competitor RNA (REN-WT 3Ј-UTR (lanes 2 and 7) or pBluescript (pBlue; lanes  3 and 8)) was added to the extracts for 30 min at 22°C before addition of labeled REN 3Ј-UTR probe. Data are representative of at least three individual experiments. B, Calu-6 extract (ϳ20 g) was incubated with either 32 P-labeled REN-WT or REN-MUT 3Ј-UTR probe, followed by RNase T1 and heparin. After UV cross-linking for 30 min, digestion with RNase A, and 10% SDS-PAGE, analysis was carried out using a PhosphorImager. 14 C-Labeled molecular mass markers are shown in lane 5. In parallel, a portion of the UV-cross-linked gel was transferred to PVDF membrane and probed with anti-HADHB and anti-HuR antibodies (*) to enable direct comparison with the RPCs. Some Calu-6 cells were treated with forskolin (FSK; lanes 2 and 7) or 4-BCA (lanes 3 and 8). "CP1" denotes an intense RPC at ϳ43 kDa.
ϳ39 kDa should provide greater insight into the REN mRNA 3Ј-UTR-multiprotein complex.
In the mitochondrial inner membrane, HADHB and very long chain acyl-CoA dehydrogenase are responsible for catalyzing the CoA esters produced by carnitine palmitoyltransferase II (51,52). HADHB is part of a multienzyme complex composed of four ␣-subunits (HADHA) with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and four ␤-subunits (HADHB) with 3-ketoacyl-CoA thiolase activity (48,49). Hydratase and dehydrogenase activities are seen in various RBPs (50,53), and our findings now implicate thiolase activity as well. Because the half-life of REN mRNA in Calu-6 cells is 4.2 h (54), a 3-fold increase in renin protein accumulation over 8 h in response to thiolase inhibition suggests that REN mRNA degradation mediated by thiolase activity may represent a significant proportion of REN mRNA turnover. Our RNAi data showing that reduction of HADHB levels leads to a marked increase in renin protein expression and a lengthening of the REN mRNA half-life are entirely consistent with the above hypothesis. Interestingly, inhibition of mitochondrial protein synthesis increases the stability of nuclear encoded mRNA transcripts, suggesting that mitochondrial proteins can affect mRNA processing (55). However, the exact mode by which this occurs is unknown.
Most RNA regulatory proteins such as HuR and CP1, although mostly nuclear, can also be present in the cytoplasm, at least transiently (56). In Calu-6 cells, however, we found HuR only in the nucleus, suggesting a role here in REN mRNA stabilization, whereas CP1 was present in both the nucleus and cytoplasm. HADHB is located on the inner mitochondrial membrane, yet our data support an ability to bind REN mRNA. In this regard, several other mitochondrial proteins can also bind mRNA. These include AUH (53), Rna14p in yeast (57), glutamate dehydrogenase (58), and 3-oxoacyl-CoA thiolase from S. peregrina (15). Taken together with our data, RNA binding appears to be a common feature of a variety of mitochondrial FIG. 5. Specific binding of HADHB, HuR, and CP1 to the REN 3-UTR. A, recombinant HADHB was incubated with 32 P-labeled REN-WT 3Ј-UTR riboprobe and, after REMSA, analyzed using a PhosphorImager. The GST control and the GST-HADHB ϩ WT probe are shown in lanes 1 and 2, respectively. In lanes 3 and 4, an ϳ100-fold excess of unlabeled (cold) competitor RNA (REN-WT 3Ј-UTR or pBluescript (pBlue)) was added to the recombinant proteins for 30 min at 22°C before addition of labeled REN-WT 3Ј-UTR and REMSA. B, recombinant HuR (GST-HuR; lanes 2, 3, and 6 -8) was incubated with 32 P-labeled REN-WT 3Ј-UTR riboprobe before REMSA. In lanes 7 and 8, an ϳ100-fold excess of unlabeled competitor RNA (REN-WT 3Ј-UTR or pBluescript) was added before addition of labeled REN-WT 3Ј-UTR and REMSA. In some lanes, anti-HuR antibody was added (GST-HuR (lane 3) and Calu-6 extracts (lane 5)). C, Calu-6 extract (lanes 1 and 2) or cleaved CP1 (lanes 4, 5, 7, 8, and 10 -12) was incubated with either 32 P-labeled REN-WT (lanes 1-6 and 10 -13) or 32 P-labeled REN-MUT (lanes 7-9), and REMSA was performed. In lanes 2, 5, and 8, anti-CP1 antibody (Ab) was added. In lanes 11 and 12, an ϳ100-fold excess of unlabeled competitor RNA (REN-WT 3Ј-UTR or pBluescript) was added as described for A. Arrows denote CP1 and RPC supershift (SS). D, shown are the results from UV cross-linking of cleaved CP1 with 32 P-labeled REN-WT (lane 1) or 32 P-labeled REN-MUT (lane 2). A component of the gel was transferred to a PVDF membrane and probed with anti-CP1 antibody using ECL (lane 4). E, shown is an immunoblot of Calu-6 cell extracts treated with forskolin (FSK); transferred to a PVDF membrane; and probed with anti-HADHB, anti-HuR, anti-CP1, or anti-actin (loading control (Con)) antibody. Actin data were similar for each (data not shown). All data are representative of at least three experiments. proteins, thus generating novel potential links between mitochondrial processes such as ␤-oxidation and mRNA metabolism. Metabolic enzymes, including glyceraldehyde-3-phosphate dehydrogenase, can bind to 3Ј-UTRs, consistent with a possible role for metabolic enzymes in RNA processing (16). Moreover, components of the citric acid cycle can control binding of IRP-1 to the transferrin receptor mRNA (59). Although mostly mitochondrial, we also saw HADHB in the cytoplasm and nucleoli. The 3-ketoacyl-CoA thiolase precursor protein is distributed equally between the cytosol and mitochondria (60), making its interaction with mRNA feasible. Studies to examine the time course of HADHB, HuR, and CP1 binding to REN mRNA, the whole cell functional consequence, and the interactions between the three proteins will provide further insight into these novel interactions.
The consequences of HADHB binding to REN mRNA in the context of the whole animal is unknown. 3-Ketoacyl-CoA thiolase is, however, within a candidate locus for blood pressure control in genetically hypertensive rat strains (63), so a link between thiolase proteins and blood pressure regulation may warrant exploration.
What could the role of HuR be in the control of REN mRNA turnover? Our RNAi data suggest a previously unrecognized important role. HuR plays a critical role in the decay of a variety of short-lived mRNAs, stabilizing them, and also has a role in nuclear-cytoplasmic shuttling (8,22,64). Recent data have clarified some aspects of HuR function. It appears to have cell-specific effects on classical AREs (65) and can undergo post-transcriptional modification (methylation) that can alter its functional capacity (66). Much less is known about the binding and functional role of HuR to less classical AU-rich cis-elements such as the SRE we found in the REN 3Ј-UTR. We have recently observed HuR binding to nonclassical ARE ciselements in the androgen receptor and p21 WAF1 mRNAs (26,27). For REN mRNA, HuR could play a critical role in mRNA stabilization in the nucleus. However, a role in facilitating transport from the nucleus to the cytoplasm for translation on ribosomes is not supported by our intracellular localization findings.
We showed previously that REN mRNA stability in Calu-6 cells is increased by forskolin (3), which activates adenylate cyclase, leading to increased cAMP. This effect is indirect, involving another gene(s). Our present data implicate HuR and CP1 in such a role, thus adding REN mRNA to the list of mRNAs that these proteins stabilize.
Our finding that CP1 co-immunopurifies and binds to the REN mRNA 3Ј-UTR adds another level of complexity to the novel RNA/protein interactions described above. The C-rich sequence in the REN mRNA 3Ј-UTR sequence (Fig. 1F) is a likely target for CP1 and spans the site of deletion in the REN-SRE mutant. This most likely explains why the REN-SRE mutant binds less well to CP1 than the wild-type probe. Interestingly, we have shown recently that HuR and CP1 also bind to the androgen receptor and p21 WAF1 mRNAs (26,27). Moreover, HuR and CP1 bind cooperatively and simultaneously to the androgen receptor cis-element (26). Thus, it will be of interest to investigate whether similar interactions exist for REN mRNA and the functional consequences of altering the expression of CP1, either alone or in combination with HuR, on renin expression. The poly(C)-binding proteins CP1 and CP2 are members of the heterogeneous nuclear ribonucleoprotein K homology domain family of RBPs (67) and regulate the stability of a variety of transcripts, including ␣-globin, tyrosine hydroxylase, and erythropoietin (68 -72), as well as translation of 15-lipoxygenase (73) and human papilloma virus type 16 E2 (74) mRNAs. Our finding that CP1 colocalizes with serine/ arginine-rich proteins in speckles adds to the repertoire of splicing and other gene expression proteins in these intranuclear reservoirs.
In conclusion, we have identified a novel set of REN mRNA-binding proteins. Remarkably, each of these proteins is well known to have important cellular functions at multiple levels. Our data point to new links between cellular thiolase activity, binding of HADHB, HuR, and CP1, and regulation of REN mRNA turnover. Finally, these novel cis-trans interactions involving the 3Ј-UTR of REN mRNA provide an array of new potential targets for therapeutics based on modulation of post-transcriptional gene expression.