Short RNA Molecules with High Binding Affinity to the KH Motif of A-Kinase Anchoring Protein 1 (AKAP1): Implications for the Regulation of Steroidogenesis
Petar N. Grozdanov and Douglas M. Stocco
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
One of the key regulators of acute steroid hormone biosynthesis in steroidogenic tissues is the ste- roidogenic acute regulatory (STAR) protein. Acute regulation of STAR production on the transcrip- tional level is mainly achieved through a cAMP-dependent mechanism, which is well understood. However, less is known about the posttranscriptional regulation of STAR synthesis, specifically the factors influencing the destiny of the Star mRNA after it leaves the nucleus. Here, we show that the 3’-untranslated region of Star mRNA interacts with the heterogeneous nuclear ribonucleoprotein K-homology (KH) motif of the mitochondrial scaffold A-kinase anchoring protein 1 (AKAP1) in vitro with a moderate affinity as measured by EMSAs. A mutation that mimics the phosphorylation state of the KH motif at a specific serine either did not alter, or had a negative impact on, protein-RNA binding under these conditions. The KH motif of AKAP1 binds short pyrimidine-rich RNA molecules with a stable hairpin structure as demonstrated by in vitro selection. AKAP1 also interacts with STAR mRNA in a dibu- tyryl-cAMP-stimulated human steroidogenic adrenocortical carcinoma cell line in vivo. Therefore, we pro- pose a model in which AKAP1 anchors Star mRNA at the mitochondria, thus stabilizing the translational complex at this organelle, a situation that might affect STAR production and steroidogenesis. In addition, we suggest that the last 216 amino acid residues of AKAP1 might participate in the degradation of STAR and other nuclear-encoded mitochondrial mRNAs through interaction with a RNA-induced silencing com- plex, specifically with the argonaute 2 protein. (Molecular Endocrinology 26: 2104-2117, 2012)
S teroid hormones regulate essential physiological pro- cesses such as reproduction, carbohydrate metabolism, and electrolyte homeostasis and are mainly produced in the gonads and adrenal glands. Deficiency in the biosynthesis of all steroid hormones results in a life-threatening condition known as lipoid congenital adrenal hyperplasia, most cases of which are caused by mutations in the steroidogenic acute regulatory (STAR) protein gene (1). STAR’s activity facili- tates the transfer of cholesterol between mitochondrial membranes to provide cholesterol substrate to the cyto- chrome P450 side-chain cleavage enzyme (P450scc; CYP11A1). Inside the mitochondria, cytochrome P450 side-
chain cleavage enzyme converts cholesterol to preg- nenolone, which is the first steroid formed in the production of all steroids (2, 3). STAR gene expression in mammals is tightly and acutely regulated by the trophic hormones of the pituitary and is mediated through cAMP-dependent mech- anisms (4-6). In addition, proper function of STAR requires type II protein kinase A (PKA)-mediated phosphorylation (7, 8), which appears to occur in the close vicinity of the mitochondria. As part of this mechanism, mitochondrial levels of PKA are elevated through its interaction with the mitochondrial scaffold A-kinase anchoring protein 1 (AKAP1, D-AKAP1) (7-11).
Abbreviations: Ago2, Argonaute 2; AKAP1, A-kinase anchoring protein 1; cv, column volume; DNase, deoxyribonuclease; KH, K-homology; IPTG, isopropyl-B-D-1-thiogalcto- pyranoside; MBP, maltose-binding protein; NFM, nonfat milk; nt, nucleotides; PKA, pro- tein kinase A; RISC, RNA-induced silencing complex; RNase, ribonuclease; SDS, sodium dodecyl sulfate; SELEX, systematic evolution of ligands by exponential enrichment; STAR, steroidogenic acute regulatory; TIS11b, tetradecandoyl phorbol acetate-inducible se- quence 11b; TOMM20, translocase of outer mitochondrial membrane 20 homolog; TSN, Tudor staphylococcal nuclease-like; UTR, untranslated region.
Proteins, like AKAP1, possessing single or multiple K-homology (KH) motifs are known to be involved in di- verse cellular actions including the synthesis of coding and noncoding RNA molecules, with the KH motif directly binding to the RNA (12-16). The RNA sequences, which usually bind with micromolar affinity to the KH motif, are comprised of several unpaired low-complexity nucleotides that interact with the hydrophobic binding pocket of the polypep- tide. Specifically, the KH motif of AKAP1 has been shown to bind to the nuclear-encoded MnSOD and Fo-f mRNAs, the products of which are located in the mitochondria (17).
The regulation of STAR gene expression has been stud- ied extensively (4, 5). However, little is known about the regulation of translation of Star mRNA or its interaction with regulatory RNA-binding proteins. Recently, it was shown that tetradecandoyl phorbol acetate-inducible se- quence 11b (TIS11b), a zinc finger protein with affinity for AU-rich RNA sequences, facilitated the turnover of STAR mRNA in a cAMP-dependent manner (18). In ad- dition, small interfering RNA-mediated knockdown of AKAP1 reduced STAR protein levels without affecting the steady-state levels of the Star mRNA (7). These find- ings suggest that AKAP1 might be involved in targeting the Star mRNA to the mitochondria and modulating the synthesis of STAR protein at its point of action. There- fore, as a first step in investigating the role of the KH motif of AKAP1 in steroidogenesis, we sought to deter- mine whether Star mRNA binds to the KH motif of AKAP1 in vivo and in vitro.
In the current study, we demonstrated that the Star mRNA associates with AKAP1 in vivo in a cAMP analog stimulated H295R human adrenocortical carcinoma cell line. We determined that the KH motif of AKAP1 interacts in vitro with the 3’-untranslated region (UTR) of the mouse Star mRNA with micromolar affinity. We also identified, by in vitro selection, unpaired pyrimidine-rich RNA sequences as the best candidates for binding to the KH motif of AKAP1. We found several of the pyrimidine-rich sequences within the 3’-UTR of the Star mRNA. In addition, we found that AKAP1, through its Tudor domain, interacts with argonaute 2 (Ago2) protein, which is part of the RNA-induced silencing complex (RISC). These findings support a model in which STAR expres- sion might be regulated on the translational level at the mito- chondrial membrane and that the localization of the Star mRNA at the mitochondria is mediated by AKAP1.
Materials and Methods
Cell culture maintenance and indirect fluorescent immunolabeling
The human adrenocortical carcinoma cell line H295R (American Type Culture Collection, Manassas, VA) was cul-
tured using the conditions that were described previously (19). Indirect immunofluorescent labeling was performed on H295R cells that were grown on size 11/2 glass coverslips (Fisher, Pitts- burgh, PA) as described previously (20). The cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA)/1x PBS for 20 min and washed twice with 1× PBS for 5 min. Subsequently, the cells were permeabilized with 1% Triton X-100/1x PBS and washed with 1× PBS, and the nonspecific binding sites were blocked with 0.5% nonfat milk (NFM)/1x PBS for 15 min. The primary rabbit antigen-purified polyclonal antihuman AKAP1 antibody (1:200 dilution; ProteinTech Inc., Chicago, IL) and monoclonal anti-translocase of outer mito- chondrial membrane 20 homolog (TOMM20) antibody (1:200 dilution; Abnova, Taipei City, Taiwan) were applied in 0.5% NFM/1× PBS for 2 h. Cells were washed three times with 1× PBS, and secondary antimouse Cy3-conjugated and antirabbit DyLight 488-conjugated antibodies, both developed in donkey (Jackson ImmunoResearch Inc., West Grove, PA), were applied in 0.5% NFM/1× PBS for 40 min. Subsequently, the cells were washed twice with 1× PBS for 5 min, stained with 4’,6-di- amidino-2-phenylindole/1× PBS for 5 min, washed once with 1× PBS, and embedded in ProLong Gold antifade reagent (In- vitrogen, Carlsbad, CA). Coverslips were cured overnight and sealed with nail polish to prevent further drying. Fluorescent and differential interference contrast images were acquired on an inverted Nikon microscope equipped with an Andor iXon EMCCD camera (Andor Technology, Belfast, UK). Images were processed for brightness and contrast using Photoshop CS2 (Adobe, San Jose, CA). Colocalization coefficients were calcu- lated using a software plug-in for ImageJ.
Construction of the expression plasmids and recombinant protein purification
Maltose-binding protein (MBP)-wild-type (WT) KH-Tudor [pPG82, the last 325 amino acid residues from the mouse AKAP1 (7), which were fused at the amino-terminal end to MBP in pPG80, which is a derivate of pMal-c possessing a PreScission protease cleavage site, a multiple cloning site, a TEV protease cleavage site, and a hexahistidine tag at the carboxy end]; MBP- S585D KH-Tudor (pPG83), similar to the WT KH-Tudor, but bearing a mutation that changes Ser585 to an aspartic acid residue (S585D); and MBP-WT KH (pPG102) and MBP-S585D KH (pPG103) spanned amino acid residues 532-645 and were created by site-directed mutagenesis using as a template pPG82 and pPG83, respectively. MBP-Tudor (pPG104) was created by site-directed mutagenesis from pPG82, removing amino acid residues from 532-642. All clones were confirmed by sequenc- ing the corresponding plasmids.
Expression and purification of recombinant proteins was performed as described previously (20, 21) with some modi- fications outlined below. The expression of the recombinant proteins was induced with 0.1 mM isopropyl ß-D-1-thiogal- ctopyranoside (IPTG) in the Rosetta 2(DE3)pLysS (EMD4 Biosciences, Darmstadt, Germany) bacterial strain. The in- duction of MBP-WT KH-Tudor and MBP-S585D KH-Tudor was carried out for 16 h at 18 C and of MBP-WT KH, MBP- S585D, and MBP-Tudor for 4 h at 37 C. The bacterial pellets (~3 g wet weight) were suspended in 50 ml buffer S3 [1.5 M NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.05% (wt/vol) NaN3, and 1 mM dithiothreitol freshly added] supplemented
with protease inhibitors (Sigma Chemical Co., St. Louis, MO), and the bacterial cells were disrupted by 15 cycles of sonication at 4 C. The insoluble material was removed by centrifugation at 15,000 × g for 20 min at 4 C. The cleared supernatants were loaded over an amylose resin (New England Biolabs, Beverly, MA) packed in a chromatographic column and preequilibrated with buffer S3. The column was washed with five column vol- umes (cv) of the buffer S3, followed by a wash with five cv of buffer S1 [0.3 M NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.05% (wt/vol) NaN3, and 1 mM dithiothreitol freshly added]. Finally, the column was washed with five cv of HEPES buffer [0.3 M NaCl, 20 mM HEPES (pH 7.6), 5 mM imidazole, 0.05% (wt/vol) NaN3] and eluted by applying five cv of HEPES buffer supplemented with 10 mM maltose. Usually, the majority of the recombinant proteins was recovered in the first half of the elu- tion volume. Recombinant proteins were further purified over a TALON (Clontech, Palo Alto, CA) resin packed in a chromato- graphic column, eluted in HEPES buffer supplemented with 200 mM imidazole. The purified proteins were concentrated using filter units with a proper membrane size (Millipore, Billerica, MA), and the buffer was exchanged to 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.05% (wt/vol) NaN3. Subsequently, the proteins were concentrated again and the molarity was deter- mined by spectrophotometry at 280 nm using the predicted molecular weight and the extinction coefficient assuming that all cysteines were reduced as calculated on the EXPASY web server (22). The molar concentration of the proteins was in the 100- to 200-UM range, except MBP-Tudor, which was 46 µM. The identity of MBP-WT KH-Tudor and MBP-S585D KH-Tu- dor was confirmed by a mass spectrometry. Before each EMSA experiment, sufficient amounts of proteins were diluted to 20 M (the highest concentration used) with serial dilutions down to 2.4 and 4.8 nM.
In vitro RNA synthesis and 3’-end fluorescein labeling
The RNAs used in the study were in vitro transcribed using a MEGAshortscript T7 kit (Ambion, Austin, TX). The DNA tem- plates for the proximal 261 nucleotides (nt) and the distal 263 nt of the 3’-UTR of Star mRNA (23), and the systematic evolution of ligands by exponential enrichment (SELEX) clones 8-1 and 8-4 were generated using PCR technology with a Pfu polymer- ase. The primers corresponding to the sense strand of the tem- plates possessed a T7 RNA polymerase promoter. DNA tem- plates were cleaned up using QIAquick PCR purification kit (QIAGEN, Valencia, CA) and ethanol precipitated. Usually, ap- proximately 8-10 µg of the DNA templates was used for the in vitro transcription reaction, which yielded 1-2 nmol RNA. Sub- sequently, the RNAs longer than 100 nt were purified using a MEGAclear kit (Ambion); RNAs shorter than 100 nt were pu- rified using RNeasy mini kit (QIAGEN), and 0.5 nmol of the purified RNAs were dephosphorylated using a combination of T4 polynucleotide kinase (New England Biolabs) and calf intes- tinal alkaline phosphatase (New England Biolabs), followed by phenol/chloroform extraction and ethanol precipitation.
The 3’-end of the RNA molecules were oxidized using so- dium periodate, and fluorescein-5-thiosemicarbazide (Invitro- gen) was used to label the RNAs with a single fluorescein dye at the 3’-end (21, 24). Labeled RNAs were cleaned up over G25 Sephadex columns (Roche Applied Science, Indianapolis, IN) to
remove the unincorporated dye. The labeling efficiency and the yield were determined by measuring the absorption at 260 and 491 nm. The efficiency of the labeling was above 80%. The labeled RNA probes were diluted in water to 100 nm before the EMSA experiments.
EMSA and determination of apparent dissociation constants (Kd,app)
For the experiments presented in Fig. 2, 300 pM 3’-end fluorescein-labeled RNA molecules were equilibrated with the serial dilution of the recombinant proteins for 3 h at room tem- perature in buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 0.01% (vol/vol) IGEPAL CA630, 5 µg/ml heparin, 10 U RNaseOUT (Invitrogen), and 10 µg/ml yeast tRNA (21). After the completion of the incubation time, the reactions were supplemented with 6% glycerol and were loaded on a 6% native vertical polyacrylamide gel in 0.5× Tris-borate/ EDTA buffer at 4 C. Experiments presented in Fig. 3 were per- formed under slightly different conditions: incubation time was shortened to 10 min at room temperature and the buffer was modified to 10 mM Tris-HCl (pH7.4), 200 mM NaCl, 0.01% (vol/vol) IGEPAL CA630, 10 µg/ml yeast tRNA, 5% glycerol. The protein-RNA complexes were resolved from the free RNA on 6% native slab polyacrylamide gels in 0.5× Tris-borate/ EDTA buffer at 4 C. No difference in the affinities was observed using either condition. The protein-RNA complexes and free RNAs were visualized using a fluorescent gel imager (Pharos FX scanner; Bio-Rad, Hercules, CA) as recommended by the manu- facturer, and the images were quantified using ImageJ software. The bound fraction of the RNA was calculated as a fraction of the bound and free RNA or as a disappearance of the free RNA and plotted toward the protein concentration. The apparent dissocia- tion constants were calculated by fitting the data to a modified version of the Hill equation (24, 25) using GraphPad Prism version 5.2 software for Windows (GraphPad Software, San Diego, CA). Unpaired t test was also performed using GraphPad Prism.
SELEX technology
SELEX technology (21, 26), with some modifications as outlined below, was used to determine high-affinity binding RNA sequences to the recombinant MBP-WT KH motif of AKAP1. To construct the initial library, 600 pmol single- stranded DNA oligonucleotide was used [N106, 5’-GCGTCA AGTCTGCAGTGAA(N)30TCGTAGATGTGAGATCCATT CCC-3’], which gave approximately 4 × 1014 different molecules. To convert the DNA oligonucleotide to double- stranded DNA, six cycles of PCR were performed with prim- ers N105 (5’-GATAATACGACTCACTATAGGGAATGGA TCTCACATCTACGA-3’) and N107 (5’-GCGTCAAGTCTG CAGTGAA-3’). The PCR product was used as a template in an in vitro transcription reaction using MEGAshortscript T7 kit (Ambion). Eight cycles of positive selection were performed. In the first two cycles approximately 60 pmol, in the second two approximately 30 pmol, in the third two approximately 15 pmol, and in the last two approximately 30 pmol of the recom- binant MBP-WT KH protein coupled to amylose beads (New England BioLabs) were incubated with approximately 0.8-1 nmol of the initial and selected RNA pools. The binding reaction was carried out at room temperature for 30 min (initial four cycles) and 10 min (last four cycles) in a binding buffer [10 mM
Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM MgCl2, 0.01% (vol/ vol) IGEPAL CA630, and 50 µg/ml yeast tRNA]. The free RNAs were separated from protein-RNA complexes by a low-speed cen- trifugation over a 45-um pore-size spin column (Millipore). The protein-RNA complexes were washed four times with binding buf- fer without IGEPAL CA630. During the last two selection cycles, salt concentration of washes two and three was increased to 500 mM NaCl. The protein-RNA complexes were eluted from the beads in the binding buffer supplemented with 10 mM maltose. The RNA was phenol/chloroform extracted and ethanol precipitated. The recovered RNA was converted to cDNA using primer N107 and Superscript III reverse transcriptase (Invitrogen). Ten cycles of PCR were performed using primers N105 and N107 and the cDNA as a template to generate a new DNA template for the next cycle of in vitro transcription, followed by selection. After the eighth cycle, a negative selection was performed, leaving out the recombinant protein, thus eliminating the RNA molecules that would be nonspecifically bound. After the negative selection, the RNA recovered from the flow- through was converted to a cDNA. The subsequent double- stranded PCR product was cloned in the pCR4-TOPO vector using the TOPO TA cloning kit (Invitrogen).
UV cross-linking, immunoprecipitation of AKAP1, Western blot, RNA purification, and RT-PCR analysis
At 80-90% confluence, three 10-cm dishes of H295R cells were stimulated with freshly prepared 1 mM dibutyryl-cAMP (Sigma) in culture media for 4 h. After completion of the incu- bation time, the treated and the same number of control cells were rinsed with 1× PBS and irradiated for 200 mJ/cm2 (UVP UV Crosslinker CL-1000 at 254 nm; UVP, Upland, CA). Cells were washed with 1× PBS, scraped in 1 ml 1× PBS, collected by low-speed centrifugation at 4 C, and frozen. The treated and control cells were lysed in 1.5 ml RIPA buffer [1× PBS, 0.5% (vol/vol) IGEPAL CA 300, 0.5% (wt/vol) sodium salt of deoxy- cholic acid, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 0.05% (wt/vol) NaN3] supplemented with SUPERaseIn (Ambion) and protease inhibitors (Sigma). The cell lysate was incubated on ice for 10 min followed by centrifugation at 20,300 × g for 20 min at 4 C. Cleared lysate was added to protein A magnetic beads (Dynabeads; Invitrogen) precoupled with 4 µg polyclonal AKAP1 antibody (ProteinTech) or 4 µg antibody against the goat IgG, matching the isotype of the AKAP1 antibody (Sigma). Immunoprecipitation was carried out for 1 h at 4 C. Magnetic beads were collected on the magnetic separator and were washed twice with RIPA buffer, followed by three washes with RIPA buffer containing five times more PBS (5× PBS), followed by two washes with RIPA buffer. To recover the RNA associ- ated with AKAP1, the immunoprecipitated AKAP1 protein was digested with 2 mg/ml proteinase K in a buffer containing 3 M urea, 0.5% (wt/vol) SDS for 30 min at 45 C, followed by phenol/ chloroform extraction and 2-propanol precipitation in the pres- ence of 0.3 M sodium acetate. Protein samples were collected for Western blot analysis at each step of the protocol. To maximize conversion of the RNA associated with the AKAP to cDNA, a reverse transcriptase reaction was performed with a combina- tion of random hexanucleotides and an oligo-deoxythymidine primer and Superscript III reverse transcriptase (Invitrogen). Primer pairs specific for the mRNA being tested were as follows:
STAR mRNA (accession number NM_000349, product size 180 bp), primers N143 (5’-CTACTCGGTTCTCGGCTGGA AGAG-3’) and N144 (5’-GCCCACATCTGGGACCACTTTA CT-3’); Fo-f mRNA (accession number NM_004889, product size 274 bp), primers N151 (5’-CAGTTGGTGAGTGTCCGGC CC-3’) and N152 (5’-TGGTATTTGCGGAGCCGCTCG-3’); MNSOD mRNA (accession number NM_000636, product size 209 bp), primers N149 (5’-ACCAGGAGGCGTTGGCCAAG- 3’) and N150 (5’-TGCAGCCGTCAGCTTCTCCT-3’); GAPDH mRNA (accession number NM_002046, product size 234 bp) primers N157 (5’-TCTTTTGCGTCGCCAGCCGAG- 3’) and N158 (5’-CCCGTTCTCAGCCTTGACGGT-3’); and MLN64 mRNA (accession number NM_006804, product size 315 bp) primers N163 (5’-CCCAGGTTGCTGTTGCCCGT- 3’) and N164 (5’-GACAGGGCAGGAAGGTCTTCAGGA-3’). PCRs were performed with EmeraldAmp GT PCR Master Mix (Clontech). Between 1/10 and one fifth of the PCRs were ana- lyzed on 2% agarose gels stained with ethidium bromide.
For immunoprecipitation, cell lysates were prepared in immunoprecipitation buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 1% (vol/vol) Triton X-100, 5% (vol/vol) glycerol] supplemented with SUPERaseIn (Ambion) and protease in- hibitors (Sigma) as previously described (27). Cleared cell lysate was added to either 4 µg isotype control antibody against the goat IgG or 4 µg antibody against AKAP1 coupled to protein A Dynabeads (Invitrogen). Immunoprecipitations with antibody against AKAP1 were either untreated or treated with ribonuclease (RNase) or RNase-free deoxyribo- nuclease (DNase) I for 5 min at room temperature followed by incubation at 4 C 1 h.
Western blots were performed using the NuPAGE SDS- PAGE gel system (Invitrogen). Primary polyclonal antibodies against AKAP1 (ProteinTech), rabbit monoclonal antibody against Ago2 (Cell Signaling Technology, Danvers, MA), mouse monoclonal anti-actin (Ambion), and secondary antirabbit and antimouse horseradish peroxidase-conjugated antibody (Thermo Scientific, Pittsburgh, PA) were consecutively applied as recommended by the suppliers. In the Western blot shown in Fig. 4A, the membrane was initially probed with anti-AKAP1 antibody, appropriate secondary horseradish peroxidase-conju- gated antibody, and developed with enhanced chemilumines- cence (PerkinElmer, Norwalk, CT). Subsequently, the same membrane was stripped in 0.1 M glycine (pH 2.6) and reprobed with anti-actin antibody.
Homology modeling and BLAST comparison
Primary protein sequence of the KH motif of AKAP1 (amino acid residues 559-637) was aligned manually to the primary sequence of the KH3 of Nova-2 protein (pdb accession number 1EC6, amino acid residues 1-81) as shown on Fig. 5A in SWISS- PdbViewer (28, 29). The alignment was submitted for a com- putational modeling on the SWISS-MODEL Workplace (30, 31). The homology model of the KH motif of AKAP1 on the KH3 of Nova-2 structure had a Z-score QMEAN of -0.82. The S585D mutation was introduced in the SWISS-PdbViewer, and all homology models were rendered in three dimensions. Phos- phate addition to the serine 585 in the homology model was kindly introduced by R. Bryan Sutton (Texas Tech University Health Sciences Center). The KH3 of Nova-2 protein demon- strated the highest percent identity to the KH motif of AKAP1 in the protein bank database.
A
AKAP1
Mito-TOMM20
AKAP1/ Mito-TOMM20
DIC
B
1 30
306319
532
857
AKAP1
M
P
KH
Tu
Identity % (Mm vs. Dr/Mm vs. Hs)
(60/97)
(35/45)
(0/79)
(35/59)
(71/96)
KH motif
Tudor
532
565
625 645
718
763
857
KH
Identity % (Mm vs. Dr/Mm vs. Hs)
(42/79)
(86/97)
(79/100)
(74/100)
(61/98)
WT KH-Tudor
KH
Tudor
S585D KH-Tudor
KH
Tudor
WT KH
KH
S585D KH
KH
Tudor
Tudor
| C | Identity (%) | |
|---|---|---|
| 532 565 625 645 | ||
| Mouse | MDSVDSCCGLTKPDSPQSVQAGSNPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQNIQICHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | |
| Human | MDSVDSCCSLKKTESFONAQAGSNPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQSVQICHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | 92% |
| Chimp | MDSVDSCCSLKKTESFONAQAGSNPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQSIQICHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | 93% |
| Rat | MDSVDSCCGLTKPDSPQTVQAGSNPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQNIQICHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | 99% |
| Dog | MDSVDGCCGPRKTDSFONAQAASSPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQNIQICHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | 92% |
| Cow | MDSVDSCCGFRKPDDFONAQAGSNPKKVDLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYTQNIQVCHIEGSQHHVDKALNLIGKKFKELNLTNIYAPPLPSL | 94% |
| Chicken | MDSVDSGCALGKTETHONSKPGGESSKSDLTIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYISTLPYSHDFQICHIEGSQHHVEKALSLIGKKFKELSLTNIYAPPPPSL | 78% |
| Zebrafish | MDSVDSCSTLGAGDGQLSASQTCQSQSSELIVWEIEVPKHLVGRLIGKQGRYVSFLKOSSGAKIYISTLPYTQEFQICHIEGTOOOVDKALALIGKKFKDLDLTNLYAPPPPPL | 72% |
FIG. 1. Subcellular localization and conserved domain organization of AKAP1. A, Wide-field fluorescent and differential interference contrast (DIC) micrographs of the human adrenocortical carcinoma cell line H295R labeled by indirect immunofluorescence with a polyclonal antibody against AKAP1 (green) and monoclonal antibody toward TOMM20 (red). In the composite image, blue represents 4’,6-diamidino- 2-phenylindole-stained nuclei. Inset, A single mitochondrion. Bar in DIC image, 10 um. B, Schematic representation of the conserved domain organization of AKAP1 as annotated on the mouse protein (GenBank accession number NP_033778). Rectangles represent the domains/motifs of the mouse AKPA1. The line represents interdomain regions. KH, Heterogeneous nuclear ribonucleoprotein KH motif; M, mitochondrial targeting sequence; P, amino acid residues that interact with PKA; Tu, Tudor domain. Below is shown the percent identity as determined by BLAST pair alignment comparison of the domains/motifs and interdomain regions between mouse (Mm) and zebrafish (Dr) and between mouse (Mm) and human (Hs) (values in bold). The secondary structure of amino acid residues 642-857 is shown as predicted by PSIPRED. Underneath is the position of the recombinant proteins used in the subsequent experiments. Black dots represent the position of the Serine 585 on the mouse KH motif and subsequent S585D mutant in the recombinant proteins. C, Amino acid residues from 532- 645 of the mouse AKAP1 aligned to several vertebrate species. The amino acid residues differing from the mouse sequence are highlighted in yellow. The dot represents serine 585 in the mouse protein.
Results
AKAP1 protein localizes to mitochondria in the human adrenocortical carcinoma cell line H295R
As shown previously by us (7) and others (32, 33), AKAP1 is localized to the mitochondria. To confirm the subcellular distribution of AKAP1 in the H259R human steroidogenic adrenocortical carcinoma cell line, double
indirect immunofluorescent labeling was performed. Vi- sual analysis of the labeling signal produced by the AKAP1 antibody showed that AKAP1 closely resembled a mitochondrial network (Fig. 1A, left) with the majority of the signal overlapping with the signal produced from the anti-TOMM20 antibody, which delineates the mito- chondria (34) (Fig. 1A). Colocalization of both signals
| Ka, app (IM) | Hill coefficient | |
|---|---|---|
| WT KH-Tudor | 0.38 ± 0.04 | 3.1 |
| S585D KH-Tudor | 0.43 ± 0.04 | 3.5 |
| WT KH | 0.75* ± 0.25 | 3.5 |
| S585D KH | 2.44* ± 0.37 | 3.5 |
A
WT KH-Tudor
S585D KH-Tudor
WT KH
S585D KH
Tudor
250
150
100
75
50
37
25
B
20 μΜ
0
2.4 nM
WT KH-Tudor
1.0-
WT KH-Tudor
000
fraction bound
0.8-
S585D KH-Tudor
ZI
*
0.6
0
2.4 nM
S585D KH-Tudor
20 μΜ
0.4-
0.2-
*
0.0
10-10
10-9
10-8
10-7
10-6
10-5
10-4
[MBP-KH-Tudor], M
C
2.4 nM
0
WT KH
20 μΜ
1.0-
WT KH
fraction bound
0.8-
S585D KH
*
0.6
2.4 nM
S585D KH
20 µM
0.4-
0
0.2-
*
0.0-
10-10
10-9
10-8
10-
10-6
10-5
10-4
[MBP-KH], M
D
0
2.4 nM
Tudor
20 μΜ
*
was confirmed with a Pearson’s coefficient of 0.835, Mander’s overlap coefficient of 0.814, and an AKAP1 to TOMM20 channel pixel ratio of 0.967. We conclude that
the majority of AKAP1 protein is associ- ated with mitochondria in the H295R adrenocortical carcinoma cell line within approximately 200 nm, the colocaliza- tion detection limit of light microscopy.
The last 325 amino acid residues from AKAP1 protein are highly conserved
BLAST algorithm comparison of the primary sequence of AKAP1 for the separate domains and interdomain re- gions showed that the last 325 amino acid residues are more conserved than the rest of the protein among vertebrate species. A more detailed analysis of the last 325 amino acid residues revealed that the most conserved region of AKAP1 was the KH motif with 86% identity between mouse and zebrafish and 97% between mouse and human (Fig. 1, B and C).
Secondary structure prediction per- formed on amino acid residues 642- 857 with PSIPRED (35) revealed that the Tudor domain and the flanking se- quences of AKAP1 closely resembled a Tudor staphylococcal nuclease-like (TSN) domain, similar to the human P100 protein (36, 37) (Fig. 1B).
The last 325 amino acid residues and the KH motif of mouse AKAP1 bind to the 3’-UTR of mouse Star mRNA in vitro
Our hypothesis suggested that AKAP1, through its KH motif, might assist in the association of Star mRNA to the outer mitochondrial membrane. To examine the ability of AKAP1 to in- teract with the Star mRNA in vitro, the following recombinant proteins, WT KH-Tudor, Tudor domain, and WT KH motif were expressed as MBP fusions and purified from bacteria. In addition, serine 585 located within the KH motif was mutated to aspartic acid (Fig. 1B, black dots; and Fig. 2, A-C, S585D KH-Tudor and S585D KH), and the corresponding recombinant proteins fused to MBP were also expressed and purified from bacteria. The substitution (S585D) is believed to
A
RNA ID
4.8 nM
WT KH
0
20 μΜ
1.0-
SR106-0
SR106-0
fraction bound
0.8-
SR106-8
*
0.6-
0
4.8 nM
WT KH
20 μΜ
0.4-
SR106-8
0.2-
*
0.0-
10-9
10-8
10-7
10-6
10-5
10-4
[MBP-KH], M
B
RNA motif I
8-4, E06, F08 H07 G07, H08 E03
F06
gggaauggaucucacaucuacgaUGGUGAGUUUUUAUUCAGCAUUCCUCUCUGuucacugcagacuugacgc gggaauggaucucacaucuacgaUGGUGAGUUUUUUAUUCAGCAUUCCUCUCUGuucacugcagacuugacgc gggaauggaucucacaucuacgaUGGUGAGUAUUUUUAUUACAGCAUUCUCUAuucacugcagacuugacgc
gggaauggaucucacaucuacgaUGGUGAAAUUCUUAUUCAGCAUUCCUUUUUCuucacugcagacuugacgc qqqaauqqaucucacaucuacqaGCUGGUGAGAUUUCUUUCAGCAUUCCUUUGuucacuqcaqacuuqacqc
RNA motif II
8-3
gggaauggaucucacaucuacgaCACAAUCUGGGUUUACCCCCAGUCUUAUGCuucacugcagacuugacgc
8-1 E10 H12 E09 E05 H04
gggaauggaucucacaucuacgaCCAUCAGUUAACACAACUGUCUUAGCUCAUuucacugcagacuugacgc gggaauggaucucacaucuacgaCCCAUCCGGACUUUCCGGUCUUAGGCCUCAuucacugcagacuugacgc gggaauggaucucacaucuacgaUUAAGCCAUCAAGCUUUAGCUUGUCUUAGCuucacugcagacuugacgc
gggaauggaucucacaucuacgaGUUACCGCUUAACCUCGUAGUCUUAGGCGUuucacugcagacuugacgc gggaauggaucucacaucuacgaUACAUUUGAAUCUUUAUCGUAGUCUUAGGCuucacugcagacuugacgc
qqqaauqqaucucacaucuacqaACUAUUUCUUCGUAGUCUUAGGCGUCUCUAuucacuqcaqacuuqacqc
C
Clones 8-4, E06, F08 -16.20 kcal/ mol
G
40A
30
10
Ģ
5’
70
3’
20
D
1.0-
8-4
0.8-
0
4.8 nM
WT KH
20 μΜ
fraction bound
0.6
8-4
0.4-
*
0.2-
0.0-
10-9
10-8
10-7
10-6
10-5
10-4
[MBP-KH], M
mimic the phosphorylation state of the KH motif. This substitution was performed because of recent reports (17, 38) that the phosphorylated KH motif at Ser585 and/or the mutant (S585D) affected the affinity of the protein for
RNA. Therefore, the impact of the muta- tion (S585D) on the affinity of the KH mo- tif for the Star mRNA under our assay and binding conditions was tested.
All the recombinant fusion proteins, except for the MBP-Tudor domain, were at least 97% pure and homogeneous as determined by Coomassie Brilliant Blue- stained SDS-PAGE (Fig. 2A). To distin- guish between protein-bound and free RNA, an EMSA (24, 25) was used. The amount of bound RNA was plotted as a function of the protein concentration. Affinities of the WT KH-Tudor and WT KH and the S585D mutants (S585D KH-Tudor and S585D KH) for the Star 3’-UTR RNA were calculated by fitting the data to the Hill equation (24, 25) (Fig. 2, B and C). All the proteins pos- sessing either WT or mutated (S585D) KH motif bound to the Star 3’-UTR RNA with affinities in the 0.38- to 2.44-UM range (see Fig. 2). We did not observe complex formation between the Tudor domain and the 3’-UTR of Star mRNA (Fig. 2D). Therefore, an appar- ent dissociation constant and a Hill coef- ficient for the Tudor domain were not calculated. Also, the recombinant pro- teins containing the Tudor domains de- graded the RNA when present in high concentrations (Fig. 2, B and D). The dif- ference in the apparent dissociation con- stants of WT KH vs. S585D KH was sig- nificant (P < 0.05).
The affinities of all recombinant pro- teins to the distal 263 nt of the 3’-UTR of Star mRNA were also determined. The values for the apparent dissociation con- stants (data not shown) were similar to those calculated for the proximal 261 nt of the 3’-UTR of Star mRNA. As ex- pected, the Tudor domain did not bind to the RNA and showed slight RNase activity. Therefore, we concluded that the KH motif of AKAP1 binds to the 3’- UTR of Star mRNA with an affinity in the nanomolar to micromolar range.
RNA selection for WT KH motif of AKAP1
Hill coefficients for affinity interactions (Fig. 2) indi- cated that the binding of the KH motif to the RNA is a
cooperative event. As a result, we hypothesized that there were multiple binding sequences within the 3’-UTR of the Star mRNA with a similar or different affinity for the KH motif of AKAP1. Therefore, an RNA selection experi- ment using a 30-nt random library was performed, and the requirements for the RNA molecule to bind to the KH motif of AKAP1 were determined.
To determine short RNA sequences that could bind with a high affinity to the WT KH motif of AKAP1, SELEX (26) was performed. Before selection, an apparent dissociation constant of 3.33 + 1 AM and a Hill coeffi- cient of 1.0 for the initial RNA pool (SR106-0) was mea- sured using EMSA (Fig. 3A). After eight rounds of selec- tion, the enriched RNA pool (SR106-8) increased its affinity for the WT KH motif by approximately 9-fold (Kd,app = 0.38 ± 0.03 µM, Hill coefficient = 0.7, Fig. 3A). At this point, the RNA pool was converted to cDNA and cloned, and individual clones were sequenced. Analysis of the sequences showed that 24% of the clones (eight of 33) could be assembled in a separate group (RNA motif I, Fig. 3B). Several of the clones in the group were represented more than once, suggesting that the SELEX enriched for RNA molecules with a high affinity for the WT KH motif. A second group (seven of 33) of sequences could also be aligned (RNA motif II), representing 21% of the clones.
Analysis of the sequences in RNA motifs I and II using the mfold RNA secondary prediction software (39) indi- cated that both motifs could form distinct stem-loop structured within the RNA molecule (up to four different structures per clone) with various low-complexity se- quences unpaired (Fig. 3B). All the RNA molecules in the RNA motif I were organized in stable secondary struc- tures with free energies ranging from -18.30 to -7.35 kcal/mol as predicted by mfold. Analysis of the structures demonstrated that the seven-base 5’-AGCAUUC-3’ con- sensus sequence was unpaired in the majority of the cases (Fig. 3C). Additional uridine-rich sequences were also un- paired (not shown), but a uniform consensus longer than three nt for these sequences could not be determined.
We did not find multiple clones with identical se- quences in RNA motif II. However, the hairpin structures (free energy ranging from -15.10 to -7.63 kcal/mol) were organized in such a way that the unique five-base (5’-UCUUA-3’) consensus sequence was unpaired. Addi- tional four- to seven-nt-long, uridine-rich sequences were also unpaired within the same structure (not shown). The five-base consensus sequence was preceded by a guanos- ine, which was invariably engaged in formation of a stem structure. In the majority of the clones, the five-base con- sensus sequence was also followed by a guanosine, which in some of the clones was involved in a formation of stems
(clone H12 in all of the structures) or was unpaired (clone H04 in all of the structures).
Analysis of the enriched RNA pool (SR106-8) showed that the free RNA existed in two discrete bands (Fig. 3A, bottom, lane 0). The top band appeared to have a stron- ger interaction with the WT KH recombinant protein. Therefore, we suggest that the RNA molecules in the up- per band form a strong secondary structure that promotes short, low-complexity sequences to remain unpaired, thus manifesting high affinity for the KH motif of AKAP1.
Next, representative clones were tested for protein- RNA binding by EMSA and the affinity for the WT KH motif of AKAP1 was determined. Both clones 8-4 (RNA motif I) and 8-1 (RNA motif II) showed similar binding patterns (Fig. 3D and data not shown). Clone 8-4 was represented three times (clones 8-4, E06, and F08) and displayed a notable apparent dissociation constant of 0.14 ± 0.04 MM. Such a Kd,app is well below the reported affinity for a single KH motif (13, 16).
RNA motif I and II sequences are present in the 3’-UTR of Star mRNA
Next, the distribution of the consensus RNA motifs, as determined by SELEX, which demonstrated a high affin- ity to the WT KH recombinant protein, were examined in the mouse and human STAR mRNA (Table 1). Query sequences were derived from the unpaired oligonucleo- tides 5’-AGCAUUCCUC-3’ and 5’-UCUUA-3’, repre- senting RNA motif I and the complete RNA motif II (Ta- ble 1). Subsequently, 24 mouse and 11 human unique sequences were mapped to the mouse and human STAR mRNA (Table 1). Analysis of the location of the mapped sequences revealed that the majority, 22 and seven, were located at the 3’-UTR of the mouse and human mRNA, respectively. Three human sequences and one mouse se- quence were positioned within the short interspersed ele- ments and the exact positions determined by the Repeat- Masker web-based software (Smit, A. F. A., R. Hubley, andP.Green, RepeatMaskerOpen-3.0,1996-2010,http:// www.repeatmasker.org). The majority of the mRNA se- quences that were mapped were between five and six nt. Only one seven-nt-long sequence (5’-CAUUCCU-3’) was mapped in the Star mRNA. Most of the sequences were pyrimidine rich. In addition, 60% of the flanking nucle- otides were pyrimidines.
AKAP1 interacts with the STAR mRNA in vivo
Having established that the KH motif of AKAP1 inter- acted with the Star mRNA in vitro, we wanted to deter- mine whether such an interaction occurs in vivo. To test this possible interaction, RNAs associated with AKAP1
| Length (nt) | Species | ||||
|---|---|---|---|---|---|
| Sequences (5'->3') | Mouse | Human | |||
| Length of mRNA (nt) | 4007 | 2695 | |||
| Accession number | NM_011485 | NM_000349 | |||
| ORF span | 57-911 | 265-1122 | |||
| Proximal 261 nt | 912-1172 | NA | |||
| Interspersed repeats | 1599-1726 (SINE/Alu) | 1792-2089 (SINE/Alu) | |||
| 1731-1943 (SINE/B2) | 2585-2695 (SINE/Alu) | ||||
| 1949-2061 (SINE/Alu) 2364-2503 (SINE/Alu) 2531-2614 (SINE/B4) 2768-2918 (SINE/B4)/C 3438-3543 (SINE/Alu)/C 3544-3570 (SINE/B4)/C 3834-3930 (SINE/Alu) | |||||
| RNA motif I | |||||
| 10 | AGCAUUCCUC | ND | ND | ||
| 9 | AGCAUUCCU | ND | ND | ||
| 8 | AGCAUUCC | ND | ND | ||
| 7 | AGCAUUC | ND | ND | ||
| 6 | AGCAUU | ND | ND | ||
| 5 | AGCAU | (C)54-58(G) | (C)994-998(C) | ||
| (C)990-994(C)b | (G)1024-1028(C) | ||||
| (C)1800-1804(C)ª | (C)1823-1827(U)ª | ||||
| (C)2098-2102(C) | |||||
| (A)2745-2749(G) | |||||
| 9 | GCAUUCCUC | ND | ND | ||
| 8 | CAUUCCUC | ND | ND | ||
| 7 | AUUCCUC | ND | ND | ||
| 6 | UUCCUC | (G)60-65 (G) | ND | ||
| 5 | UCCUC | (U)61-65 (G) | (C)1573-1577(C) | ||
| (C)1297-1301(U) | |||||
| 8 | GCAUUCCU | ND | ND | ||
| 7 | GCAUUCC | ND | ND | ||
| 6 | GCAUUC | (G)3323-3328(U) | ND | ||
| 5 | GCAUU | (G)3323-3327(C) | (A)1824-1828(U)ª | ||
| (G)3562-3566(U)ª | |||||
| (U)3786-3790(A) | |||||
| 7 | CAUUCCU | (A)2314-2319(G) | ND | ||
| 6 | CAUUCC | (A)2313-2318(U) | ND | ||
| 5 | CAUUC | (A)2313-2317(C) | (A)278-282(A) | ||
| (U)2738-2742(U) | (A)755-759 (A) | ||||
| (G)3324-3328(U) | (U)1336-1340(A) | ||||
| (A)3387-3391(A) (U)3467-3471(U)ª (U)3746-3750(U) | |||||
| (U)4003-4007 | |||||
| 6 | AUUCCU | (C)2315-2319(G) (A)2686-2691(U) | ND | ||
| 5 | AUUCC | (G)181-185(A) | (A)2438-2442(A) | ||
| (U) 1354-1358(A) | (A)2550-2554(C) | ||||
| (C)2314-2318(U) | |||||
| (A)2686-2690(U) | |||||
| 5 | UUCCU | (G)60-64(C) | (C)34-38(U) | ||
| (U) 1571-1575(A) | |||||
| RNA motif II | (C)2238-2242(U) (A)2316-2319(G) (A)2687-2691(U) | ||||
| 5 | UCUUA | (G)1216-1220(C) (G)3019-3023(A) (A)3292-3296(U) | (U)2275-2279(A) | ||
Sequences derived from the clone 8-4 and RNA motif II are shown in uppercase letters. In the interspersed repeats, C represents the sequence corresponding to the complementary consensus sequence of the interspersed repeat. The positions of the unique sequences are in bold. Overlapping sequences are shown in normal text. Sequences within the open reading frame (ORF) of STAR mRNA are underlined. Nucleotides in front of and following the consensus in mouse and human STAR mRNA are shown in parentheses. NA, Not applicable; ND, not determined; SINE, short interspersed elements.
a Sequences within the short interspersed elements.
b Sequence presented in the proximal 261 nt of mouse Star mRNA.
A
Input FIAK FI IgG IP AK IP IgG -+ + -+ -+ +
B
IP AK IP IgG
dbcAMP
- + - + C
500
400
STAR, 30 cycles
250-
300
150-
200
100-
100
IP AK IP IgG - + - + C
75-
500
MNSOD, 30 cycles
+
C
400
500
400
ATP5J2, 30 cycles
300
300
50-
200
200
100
37-
1
2
a: AKAP1
3
4
5
6
7
8
9
MLN64, 33 cycles
+
C
500
500
GAPDH, 30 cycles
+
C
50-
400
400
300
300
200
200
37-
α: B-actin
100
100
C
Input
FI IgG
FI AK
IP IgG
IP AK
D
Lanes
α: Ago2
250-
Top
Bottom
150-
IP: Control isotype IgG
100-
6
75-
IP: AKAP1 IgG
1
2
3
4
5
6
7
8
9
a: AKAP1
7
250-
Top
IP: AKAP1 IgG
+RNAse
150-
8
100-
75-
IP: AKAP1 IgG
+DNAse
Bottom
9
1
2
3
4
5
6
7
8
9
α: Ago2
were UV cross-linked to the protein. Subsequently, AKAP1 and associated RNAs were recovered by immu- noprecipitation of the AKAP1 under stringent washing conditions permitting only the RNA molecules with covalent bonds to the protein to be recovered. This experiment was performed in the steroidogenic H295R
human cell line with and without treatment with 1 mM dibutyryl- cAMP, which stimulates the expres- sion of STAR (19). Western blot anal- ysis, using an antibody against AKAP1, of a total cell lysate from UV-irradiated H295R cells revealed two bands with an apparent molecular mass of approxi- mately 140 and 52 kDa (Fig. 4A, lanes 1 and 2, input). The 140-kDa band was likely AKAP1 (9). However, the identity of the approximately 52-kDa band was not clear, because the band was not pres- ent in total protein samples from human Hela cells (data not shown). Stimulation of the H295R cells for 4 h with 1 mM dibutyryl-cAMP slightly elevated the amount of AKAP1 over the basal level (Fig. 4A, lane 1 vs. 2). Immunoprecipita- tion with the anti-AKAP1 antibody al- most completely depleted AKAP1 from the flow-through, but it did not change the amount of the unidentified approxi- mately 52-kDa protein (compare lanes 1 and 2 vs. lanes 3 and 4; Fl AK). The ma- jority of AKAP1 was observed in the im- munoprecipitated samples (lanes 7 and 8). A control antibody did not precipi- tate AKAP1 or the unidentified protein (lane 9). These results suggested that the anti-AKAP1 antibody efficiently precip- itated AKAP1 from H295R cell line.
After the immunoprecipitation, RT- PCR was performed. The STAR mRNA associated with AKAP1 was consistently detected above the background upon stim- ulation with 1 mM dibutyryl-cAMP (Fig. 4B, lanes IP AK +), was not detected in the H295R cells that were not tre- ated with 1 mM dibutyryl-cAMP (lane IP AK -). The mRNAs of the manganese superoxide dismutase (MNSOD) and F2 subunit of mitochondrial ATP synthase (ATP5J2 and Fo-f) were also consis- tently detected. These mRNAs have been previously shown to be nuclear-encoded, integral mitochondrial proteins that interact with the KH motif of AKAP1 in vitro (17). The amount of the PCR prod- ucts for the MNSOD and ATP5J2 were almost identical in both dibutyryl-cAMP-treated and control samples. Two mRNAs that have not been shown to interact with the AKAP1, the metastatic lymph node 64 protein (MLN64)
mRNA and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were also identified. This suggests a di- rect interaction between AKAP1 and the STAR mRNA in vivo.
AKAP1 interacts with Ago2 protein in the H295R cell line
We established that the AKAP1 possesses a Tudor/ TSN domain using secondary structure prediction soft- ware (Fig. 1B). Therefore, we tested whether Ago2 pro- tein, part of the RISC reported to be associated with the mitochondria (40), interacts with AKAP1 using an im- munoprecipitation protocol against the endogenous proteins.
Cleared cell lysates from H295R cells were subjected to immunoprecipitation with either isotype control anti- body against goat IgG or AKAP1 antibody (Fig. 4C). As expected, the control IgG did not deplete the AKAP1 from the cell lysate (Fig. 4C, lane 2). However, the antibody against AKAP1 completely removes the AKAP1 protein from the cell lysate (Fig. 4C, lanes 3-5). Similarly, the immunoprecipitated samples revealed that the isotype control antibody does not immunoprecipitate, but the AKAP1 antibody does precipitate the AKAP1 (Fig. 4C, lanes 6-9). In addition, because both AKAP1 and Ago2 protein interact with RNA, the role of nucleic acid, which may mediate the interaction between the proteins was evaluated. Samples were treated with RNase (Fig. 4C, lanes 4 and 8), and RNase-free DNase I (Fig. 4C, lanes 5 and 9). The treatment with either enzyme does not change the profile of the immunoprecipitated AKAP1 (Fig. 4C, lanes 7-9). The same set of samples was probed with an antibody toward Ago2 protein (Fig. 4, C and D). The control background antibody (Fig. 4C, lane 6, and Fig. 4D) does not pull down the Ago2 protein. However, AKAP1 antibody efficiently pulls down the Ago2 protein with or without treatment with RNase or DNase I (Fig. 4C, lanes 7-9, and Fig. 4D). However, the flow-through fractions depleted of AKAP1 do not show diminished levels of Ago2 protein, suggesting that the AKAP1 inter- acts only with a small fraction of Ago2 pool of proteins (Fig. 4C, lanes 2-5).
Discussion
One of the objectives of the current studies was to deter- mine whether there existed an interaction between STAR mRNA and the KH motif of AKAP1 in vitro and in vivo. A second objective was, if such an interaction occurs, to identify the RNA sequences that bind the KH motif. Es- tablishing such an interaction would support a model for
the localization and translation of nuclear-encoded STAR mRNA and other integral mitochondrial proteins in both general and specific cellular processes, in this case, the initial steps of steroidogenesis.
Our data demonstrate that the KH motif and the last 325 amino acid residues of AKAP1 bind to the 3’-UTR of STAR mRNA in vitro with binding affinities that are comparable to those previously reported for single KH motifs (13, 16). We determined that unpaired pyrimidine- rich RNA sequences bind to the KH motif of AKAP1. However, the primary RNA sequences are not sufficient for optimal binding, and a stable secondary structure is required. Paradoxically, most of the sequences within the 3’-UTR of STAR mRNA identified as potential binding sites for the KH motif of AKAP1 (Table 1) are not un- paired but are involved in the formation of the stem segment of the secondary structure that may hinder the interaction. However, the five-nt-long 5’-UCUUA-3’ sequence located at 3019-3023 of the mouse 3’-UTR of Star mRNA is unpaired. This sequence is located in close vicinity to the AU-rich element previously identi- fied to be involved in the down-regulation of Star mRNA expression through its association with TIS11b (18). These findings suggest that multiple protein factors may participate in the modulation of the binding of Star mRNA to the KH motif of AKAP1 in vivo either through direct interaction with the binding sites or through mod- ulation of the secondary structure of the mRNA.
It has been reported (17, 38) that the phosphorylation of Ser585 located at the consensus PKA binding site (R582YVS585) within the KH motif of AKAP1 increases the affinity of the motif for RNA. However, our results do not support this observation (Fig. 2). Homology model- ing of the KH motif of AKAP1 on the crystal structure of the KH3 motif of Nova-2 protein (14) (Fig. 5) agrees with our experimental observation that the S585D mutant has a negative or inconsequential impact on RNA binding. The homology model reveals that Ser585 is located in the middle of the second a-helix of the motif and is likely involved in the formation of the nucleic acid-binding hy- drophobic groove. The phosphorylation of the Ser585 would 1) alter the electrostatic potential of the binding pocket, which would hinder the interactions with the neg- atively charged nucleic acid, and 2) impose steric con- straints on the binding of the nucleic acid (Fig. 5B). Sim- ilar conclusions, albeit to a smaller extent, are also valid for the S585D mutant of the KH motif (Fig. 5B). How- ever, we do not exclude the possibility that S585D sub- stitution does not act as a phosphomimetic mutant, which might explain the similar binding affinity for the WT and S585D mutant proteins.
Nova-2 KH3
A
ß1
α1
a2
B2
₿3
a3
565
625
AKAP 1
KH
DLIIWEIEVPKHLVGRLIGKQGRYVSFLKQTSGAKIYIST
LPYTQNIQICHIEGSQHHVDKALNLIGKKFKELNLTNI
+ EI VP++LVG ++GK G+ +
++ +GA+I IS
LP T+N ++ I GS
A LI ++
MKELVEIAVPENLVGAILGKGGKTLVEYQELTGARIQISKKGEFLPGTRNRRVT-ITGSPAATQAAQYLISQRVTYEQGVRA
B
WT
S585D
WT-Phos
We also determined that AKAP1, through its Tudor/TSN domain and Ago2, which is part of a complex known as RISC, form a direct protein-protein interaction (Fig. 4C). This interaction is not mediated by nucleic acid (Figs. 4, C and D). AKAP1 may either interact with cellular P-bodies, reported recently to be transiently associated with mito- chondria (41), or with the Ago2 protein, which is directly associated with the outer mitochondrial membrane (40), or both. This finding further supports the bioinformatic obser- vation that the Tudor domain of AKAP1 is likely organized as a TSN domain. The EMSA results using the purified MBP-Tudor domain of AKAP1 also indicated that the Tu- dor/TSN domain possessed weak RNase activity (Fig. 2, B and D), as previously observed for an Escherichia coli- expressed and purified TSN domain (42). In addition, a recent article demonstrates that natural antisense transcripts of the Star mRNA are involved in the regulation of STAR expression (43). Therefore, degradation of Star mRNA by the RISC coupled with the involvement of the Tudor/TSN domain of AKAP1 would ensure that a single mRNA is translated only once and is immediately degraded. By these actions, an acute and precise regulation of the synthesis of steroid hormones would be achieved and would agree with many previous observations, including those that demon- strated the need for ongoing transcription and translation in
the maintenance of steroidogenesis. The exact nature of such a process would require further experimentation.
We would like to propose the following cotranslational import model for STAR expression, a scheme that has been described for other nuclear-encoded mitochondrial protein mRNAs as reported by other investigators including Ahmed and Fisher (44), Saint-Georges et al. (45), Garcia et al. (46, 47), and Eliyahu et al. (48). Those investigators presented models in which nuclear-encoded mitochondrial proteins could be either posttranslationally or cotranslationally im- ported into mitochondria. Slightly more than half of nucle- ar-encoded mitochondrial proteins are cotranslationally im- ported. Using the cotranslational model, nuclear-encoded mRNA having proteins localized to the mitochondria can be segregated into two different groups: 1) mRNAs localizing to the close vicinity of the mitochondria and requiring inter- action with mitochondrial anchored RNA-binding protein and 2) mRNAs colocalizing at the mitochondria with- out requiring mediation by an RNA-binding protein. In this study, we show that the KH motif of AKAP1 in- teracts with pyrimidine-rich sequences and possesses affinity for the 3’-UTR of Star mRNA in vitro and interacts with it in vivo. Previous observations from our laboratory demonstrate that small interfering RNA-mediated knockdown of AKAP1 abrogated STAR
protein synthesis (7) but did not alter the steady-state level of the Star mRNA. This suggests that one of AKAP1’s functions is to aid in the efficient expression of the STAR protein. Because the steady-state level of Star mRNA does not change after AKAP1 knockdown, it is plausible that AKAP1, through its KH motif, tethers Star mRNA at the outer mitochondrial membrane. More than one AKAP1 protein may be involved in this action, because the KH motif recognizes low-complexity RNA sequences. Given our current results, we favor the cotranslational mecha- nism for STAR protein import that involves the use of AKAP1 as a mitochondrial scaffold protein anchoring the STAR mRNA to the outer mitochondrial membrane.
However, we are aware that at present we lack observa- tions that unequivocally demonstrate the localization of the Star mRNA at the mitochondria under conditions that pre- serve the morphology of the cell, e.g. fluorescent in situ hy- bridization. To our knowledge, this relationship between Star mRNA and mitochondria has not yet been shown. Our own attempts to measure the distance between Star mRNA and mitochondria using double fluorescent in situ hybrid- ization were not successful, despite using a previously published protocol (49) designed for making such measurements.
This proposed model provides an additional, and some- what unique, mechanism for controlling the acute synthesis of steroid hormones, with that control being at the level of translation of the STAR protein in the vicinity of the mito- chondria. In general, it is believed that once nuclear-encoded mitochondrial protein mRNAs are transcribed, they will quickly and automatically be translated and the proteins imported into the mitochondria. Several reports (48, 50-52) demonstrate that more than half of the nuclear-encoded mi- tochondrial mRNAs in yeast are localized in the close prox- imity of the outer mitochondrial membrane. This strongly suggests that an additional posttranscriptional step regulat- ing the expression of mitochondrial proteins exists. This process may involve the translocase complex of the mito- chondria as well as additional RNA-binding proteins that have been shown to further influence the localization, sta- bility, and translation of the transcripts of the majority of the nuclear-encoded mitochondrial mRNAs at the mitochon- dria (53). It is possible that STAR expression, and thus ste- roidogenesis, is also influenced by this mechanism.
Acknowledgments
We thank Drs. Jeffrey A. Chao, Matthew T. Dyson, Luchezar K. Karagyozov, Steven R. King, Clinton C. MacDonald, and R. Bryan Sutton for helpful discussions. In addition, we thank Drs. Chao and MacDonald for valuable suggestions on the manuscript. We also
thank Kerry Fuson and Kamakshi Nayak for technical support in the protein buffer exchange. We thank Dr. Barry Maurer for the use of the Pharos FX scanner and Dr. Sutton for introducing the phosphate group at the Ser585 of the homology model of the KH motif. We also thank Dr. Raul Martínez-Zaguilán for help with the microscopic imaging.
Address all correspondence and requests for reprints to: Douglas M. Stocco, Department of Cell Biology and Bio- chemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. E-mail: doug.stocco@ttuhsc.edu.
This work was supported by National Institutes of Health Grant HD-17481 and with funds from the Robert A. Welch Foundation (Grant B1-0028).
Disclosure Summary: The authors have no disclosures to make.
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