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Department of Pharmacology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, Wisconsin-Madison 53706, USA
1 Molecular and Cellular Pharmacology Graduate Program, University of Wisconsin-Madison, Madison, USA
(Requests for offprints should be addressed to C R Jefcoate; Email: jefcoate{at}wisc.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The acute stimulation phase involves regulation of both mRNA levels and several additional processes. Synthesized as a 37 kDa precursor (p37), StAR is imported into mitochondria where its leader sequence is cleaved by mitochondrial proteases to generate a series of 28–30 kDa forms. The C-terminus of StAR harbors a cholesterol-binding START domain (Tsujishita & Hurley 2000). Studies using non-steroidogenic COS-1 cells indicate that StAR can function at the outer mitochondrial membrane (Arakane et al. 1996). However, in much more active steroidogenic cells, StAR needs to be continually translated and imported into mitochondria to be optimally effective (Ferguson 1963, Privalle et al. 1983, Epstein & Orme-Johnson 1991, Stocco & Sodeman 1991, King et al. 1999, Artemenko et al. 2001, Bose et al. 2002). Functionally disruptive mutations of StAR gene cause the congenital lipoid adrenal hyperplasia disease in human (Fujieda et al. 2003), and similar phenotypes are observed in StAR null mice (Caron et al. 1997). Several other proteins have also been implicated in cholesterol delivery to the inner membrane cytochrome P450scc. These include the peripheral-type benzodiazepine receptor (PBR), an interacting protein PAP7, an additional StAR-binding protein and enzymes involved in fatty acid delivery to mitochondria (Liu et al. 2003, Sugawara et al. 2003, Cornejo Maciel et al. 2005, Hauet et al. 2005). Processes involved in the delivery of cholesterol to mitochondria are also critical to the activation of steroidogenesis. These include activation of hormone-sensitive lipase and suppression of acyl CoA: cholesterol acyltransferase (ACAT) by tropic hormones (DiBartolomeis & Jefcoate 1984, Temel et al. 1997, Buhman et al. 2000, Kraemer et al. 2004).
In mouse and rat steroidogenic cells, two predominant forms of StAR transcripts of 1.6 and 3.5 kb are expressed (Clark et al. 1995). A minor 2.8 kb form reported earlier was not consistently detected in our experiments. We have shown that the two major transcripts arise from alternative use of polyadenylation signals in exon 7 (Ariyoshi et al. 1998). The two transcripts share the same 5'UTR and open reading frame, only differing in their 3'UTR. The 3.5 kb transcript has an extended 3'UTR not present in the 1.6 kb transcript (Fig. 1
). Several AUUUA motifs are found at the 3' end of the 3.5 kb transcript. In an earlier publication, we found that the two messages are regulated differently by mRNA stability mechanisms through their 3'UTRs (Zhao et al. 2005). The abundance of an mRNA species is determined by multiple processes, including transcription, pre-mRNA processing, nuclear export, and cytoplasmic turnover. In particular, transcript stability is an important control point of its expression level. The half-lives of mammalian mRNAs range from < 20 min to over 24 h, and can readily be altered by external stimuli. An increase in the stability of a labile mRNA can provide a faster response than transcriptional regulation.
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Regulation of mRNA stability provides yet another mechanism that also addresses the independent regulation of the different StAR transcripts. Sequence elements that stabilize or destabilize a transcript can occur in its 5'UTR, open reading frame, or 3'UTR (Guhaniyogi & Brewer 2001). Among the best studied and most prevalent of these is the AU-rich element (AURE), present in the 3'UTR of many labile cytokine and proto-oncogene mRNAs. Several classes of AURE have been described, which contain clustered or distributed AUUUA pentameric motifs or a U-rich sequence (Brennan & Steitz 2001). AURE mediate rapid turnover of transcripts as well as their stabilization or destabilization by signal transduction pathways. Multiple AURE-interacting proteins with opposing effects on mRNA stability have been identified, such as AUF-1, TTP, KSRP (destabilizing), and HuR (stabilizing). These factors function by either recruiting mRNA metabolizing machineries or protecting the message from enzymatic degradation (Bevilacqua et al. 2003). Signal transduction pathways change decay rates of AURE-containing transcripts through altering binding properties of AURE-interacting proteins (Wilusz & Wilusz 2004).
In addition to the cytoplasmic stability regulation, the decay of nuclear RNA is an important and highly regulated process for ensuring mature mRNA quality (Moore 2002). Inefficiently processed, unpolyadenylated or erroneously transcribed messages are targeted for degradation by the nuclear exosome (Vinciguerra & Stutz 2004). Introns also impact the nuclear retention and subsequent decay of nascent transcripts (Le Hir et al. 2003). The packaging of ribonucleoprotein complexes is initiated on intron sequences, thus enhancing the possibility of degradation of simple cDNA transcripts.
The majority of genes that are controlled at the mRNA stability level are needed for acute cellular responses to stimuli, such as early response genes, cytokines, and inflammatory mediators. The predominance of the less stable 3.5 kb StAR provides the opportunity for similar acute regulations. A key feature of regulation through mRNA stability is that the mRNA is unstable under basal conditions such that stabilization enhances its expression. In this paper, we show that the 3.5 kb mRNA provides this basal instability and resolve contributions of the 3'UTR sequence elements that are responsible for differential regulation of basal stability of StAR transcripts. To this end, StAR and luciferase expression vectors with short or long 3'UTR sequences were constructed. We used these vectors to separate non-specific and sequence-specific effects on StAR mRNA and protein expression levels within the 3'UTR. We also introduced mutations into AURE motifs to elucidate their contributions. Comparisons of StAR and luciferase chimeras were used to check the effects of StAR 5'UTR and open reading frame (ORF). These effects were examined in non-steroidogenic COS-1 cells and in steroidogenic Y-1 and MA-10 cells. We employed different approaches to study StAR mRNA stability, based on steady-state expression from cytomegalovirus (CMV) promoter constructs and direct measurement of transfected mRNA half-life. The steady-state differences in protein and mRNA expression caused by the extended 3'UTR were far greater than the differences in mRNA stability. To further examine whether this effect of 3'UTR was related to post-transcriptional nuclear events, we used isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible luciferase chimeras to measure the effect of 3'UTR on kinetics of luciferase appearance in the cytoplasm. The well-established role of introns in nuclear events also led us to compare the expression of StAR from a full length construct with or without the six introns.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chemicals were obtained from Sigma Chemical Company at the highest grade unless otherwise stated. Cell culture media and horse serum were bought from Invitrogen/GIBCO Company. Fetal bovine serum was purchased through Atlanta Biologicals, Inc. (Lawrence-ville, GA, USA). The TransIt-LT1 reagent for DNA vector transfection and TransIt-mRNA kit for mRNA transfection were from Mirus Bio Corporation (Madison, WI, USA). Pfu Ultra enzyme for PCR cloning was purchased from Stratagene (La Jolla, CA, USA). Restriction enzymes were purchased from Promega. Plasmid and RNA preparation kits were purchased from Qiagen. Cell culture flasks, dishes, and plates were purchased from Corning, Inc. (Corning, NY, USA).
Plasmid constructs
Primers for cloning were all designed with a 5' overhang of four to six bases, followed by the appropriate restriction sites, and a 25–30 base region that complements the original vector at the 3' end.
Rat and mouse StAR expression constructs are derived from the pCINeo mammalian expression vector (Promega) driven by a CMV promoter. The 1.6 and 3.5 kb rat StAR constructs were described previously (Zhao et al. 2005). To make the StAR construct without the 3'UTR (StARdUTR, Fig. 2A), the primer pair 5'-AGCTCTCGAGGA ACAAATCCCT GGGAGCA G-3' and 5'-GGTGTCTAGACCTTAACACTGGGCCTCA GA-3' were used to amplify StAR3.5k vector. The PCR product was cut with XhoI/XbaI and ligated into pCINeo. Mouse StAR vectors mStARdUTR, mStAR1.6k, and mStAR3.5k were made by amplifying mouse cDNA (isolated from Y-1 cells) with the forward primer 5'-TA GTCTAGATCAGGACCTTGAAAGGCTCAGG-3' and reverse primers 5'-GTGGCGGCCGCTTAACACTGGG CCTCAGAGGCA-3', 5'-CTGGCGGCCGCATGTATGTT CTTTATTGTCATG-3' and 5'-TAGGCGGCCGCATGCC AGTGACAACTGCTTTTA-3' respectively. PCR products were digested by XbaI/NotI and ligated into the XbaI/NotI sites of pCINeo. To make mStAR3.5/introns vector, the primers 5'-CAGCACGCGTACTCAGGACC TTGAAAGGCTCAGGA-3' and 5'-CCCATCTAGAGAAT-GACTATTAACACAATTTAATA-3' were used to do long-range amplification of mouse genomic DNA (isolated from Y-1 cells). The resulting 7.5 kb mouse StAR gene was digested with MluI/XbaI and ligated into the MluI/XbaI sites of pCINeo.
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), the primers 5'-CCAGAC-TAGTGGACTGCCCACCACATCTAC-3' and 5'-TTAG-ACTAGTGTCTATTAACGTCAAATGAC-3' were used to amplify StAR3.5k vector; for UTRL and UTRLas, 5'-CC AGACTAGTGGACTGCCCACCACATCTAC-3' and 5'--TGTCACTAGTACCTATAATCAGACTGCCAAT-3' were used; for UTRdARE, 5'-CCAGACTAGTGGACTGCCC ACCACATCTAC-3' and 5'-TCATACTAGTGCGC AGTCCTTGCAGGTGAT-3' were used; for vector 684-2115 and 684-2115as (Fig. 4A), 5'-AGAACTAGT CCTAAAAATGTAATTTCATAACAAACAC-3' and 5'-GA CACTAGTCTCGGAGGGACAGAAAAGTGGGCCA-3' were used. PCR products were digested with SpeI and ligated to the XbaI site after luc+. Insert directions were determined by sequencing.
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) was generated using the primers 5'-CCACTGCAGTTACACGGCGATCTTTCC GCCCTTC-3' and 5'-CCCCTGCAGTAAACGGTCTTA GTCGTCTGGGTCC-3' to amplify UTRL, after which the PCR product was digested with PstI and self-ligated. Constructs 684–1043, 684–1403, and 684–1763 (Fig. 6A) were made by amplifying 684–2115 using the forward primer 5'-AGACTGCAGTCGGGGCGGCCGGCCG-CTTCGAGC-3' and reverse primers 5'-GGACTGC-AGTGTCCACTAAGGCTACAATAGGGCA-3', 5'-GAC-CTGCAGTCTGTTACCAACTGACCTCATAAAC-3', 5'-TCTCTGCAGCTTCCTGGAGTCTTCCCTCTTTGTG-3' respectively. PCR products using these primer pairs were digested with PstI and self-ligated. The vector 1043–1763 was made by amplifying 684–1763 using primers 5'-TAG-TCTAGACCAGGCTCCCACATGGTACACAGAC-3' and 5'-AGGTCTAGAATTACACGGCGATCTTTCCGCCCTT-3'. PCR product was digested with XbaI and self-ligated. The vector 1043–1763as was made by amplifying 684–1763 using primers 5'-TAATCTGCAGCCAGGCTCCCA-CATGGTACACAG-3' and 5'-CCGATCTAGACTTC-CTGGAGTCTTCCCTCTTTG-3'. PCR product was digested with PstI/XbaI and ligated into PstI/XbaI digested 684–1763 vector backbone.
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IPTG-inducible luciferase vectors were made by introducing inserts into pOPRSVI/MCS (Stratagene). For LacLuc (Fig. 7B), the primers 5'-TGGCTCGAGGG TACTGTTGGTAAAGCCACC-3' and 5'-GACTCTAGAATTACACGGCGATCTT-3' were used to amplify the pG L3-promoter vector. PCR product was digested with XhoI/XbaI and ligated to the XhoI/XbaI sites within pOPRSVI/MCS. For LacLuc–UTRS, 5'-TGGCTCGAGGG TACTGTTGGTAAAGCCACC-3' and 5'-CCCGCGGCCG CTGTCTATTAACGTCAAATGAC-3' were used to amplify UTRS; for LacLuc–UTRL, 5'-TGGCTCGAGGGTAC TGTTGGTAAAGCCACC-3' and 5'-CCCGCGGCCGC TACCTATAATCAGACTGCCAAT-3' were used to amplify UTRL. PCR products were digested by XhoI/NotI and ligated to the XhoI/NotI sites within pOPRSVI/MCS.
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Cell culture and DNA vector transfection
Y-1 mouse adrenocortical tumor cells were expanded from a subclone obtained from Dr Bernard Schimmer (University of Toronto) that has a lower passage number than those available from ATCC. They were cultured in F-12K media (Sigma) supplemented with 15% horse serum, 2.5% fetal bovine serum, 17.86 mM NaHCO3, 50 IU penicillin, and 50 µg/ml streptomycin. MA-10 mouse Leydig tumor cells were a generous gift from Dr Mario Ascoli (University of Iowa College of Medicine). They were maintained in D-MEM/F-12 media (GIBCO) supplemented with 5% horse serum, 2.5% fetal bovine serum, 26.66 mM NaHCO3, and 50 µg/ml gentamicin. To ensure consistent good growth, MA-10 cells were cultured on 0.1% gelatin-coated plates (Hirakawa et al. 2002). COS-1 cells were purchased from ATCC and cultured in D-MEM with high glucose, L-glutamine, and sodium pyruvate (GIBCO), supplemented with 10% fetal bovine serum, 17.86 mM NaHCO3, 50 IU penicillin, and 50 µg/ml streptomycin. All cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
StAR and luciferase expression vectors were transfected with TransIt-LT1 (Mirus Bio Corp.) according to manufacturer’s protocol. Briefly, cells were seeded in 24- or 6-well plates at 25% density 24 h prior to transfection. Triplicate cultures were transfected with the same molar amounts of each DNA vector (calculated by base pairs) at around 400 ng/well in 24-well plates, together with 40 ng/well of pRLTK control vector (Promega) for dual-luciferase cotransfection where applicable. This was scaled up accordingly for six-well plates. Ratio of total DNA:TransIt-LT1 reagent:OPTI-MEMI media is 1 µg:2.5 µl:50 µl. OPTI-MEMI media (GIBCO) were first mixed with TransIt-LT1 and incubated for 15 min at room temperature. DNA vectors were then added, mixed thoroughly, and incubated for another 15 min. This transfection media mixture was directly aliquoted to cells cultured in complete media with serum and incubated for 24 h. For luciferase activity measurements, cells were harvested in 1x passive lysis buffer and assayed using Promega’s Dual-Luciferase kit on a Pharmingen Mono-light 3010 luminometer per manufacturers’ instructions.
Data from luciferase transfections are expressed as means ± S.D. calculated from the triplicate cultures.
Site-directed mutagenesis
The first, second, and the third UUAUUUAU motifs within rat StAR AURE were mutated to UUCGUUAU using the following primer pairs.
First motif 5'-CCTGCAAGGACTGCGCTTCGTTAT-GAACAGAACAACGT-3' and 5'-ACGTTGTTCTGTTCATAACGAAGCGC AGTCCTTGCAGG-3'
Second motif 5'-CAACGTGGAACGCGTGTTTCGTTATTGAAGTCTGAAGACT-3' and 5'-AGTCTTCAGACTTCAATAACGAAACACGCGTTCCACGTTG-3'
Third motif 5'-TCTGAAGACTTAACAGCTGGTCGTTATTGTAATTCATTCTGACT-3' and 5'-AGTCAGAATGAATTACAATAACGACCAGCTGTTAAGTCTTCAGA-3'
Mutagenesis was performed using Pfu-Ultra enzyme (Stratagene) to amplify the wild-type ARE vector. PCR cycling parameters were: 30 s at 95 °C, then 18 cycles of (30 s at 95 °C, 1 min at 55 °C, 9 min at 68 °C). PCR products were extensively digested with DpnI enzyme and transformed into JM109 Escherichia coli competent cells. Mutant vectors were identified by sequencing.
In vitro transcription and mRNA transfection
In vitro transcription vectors for rat StARdUTR, 1.6 and 3.5 kb mRNAs were made as described previously. They were linearized with HindIII enzyme and transcribed using Ambion’s mMessage mMachine T7 kit per manufacturer’s protocol. The kit contains a 5'mRNA cap analog and capping enzyme activity. Transcribed messages thus is complete with a functional 5' cap as well as 90-base poly(A) tail from the vector. Messages were checked on a denaturing agarose gel to confirm uniformly correct sizes.
mRNAs were transfected using Mirus Bio’s TransIt-mRNA transfection kit. Cells were plated in 12-well plates at 25% density 24 h prior to transfection. One microgram mRNA was transfected in each well. The following proportion was used: 1 µg mRNA, 1 µl TransIt-mRNA reagent, 1 µl mRNA boost reagent, and 100 µl serum-free media. mRNA was first mixed with serum-free media, after which mRNA boost reagent was added and mixed, followed immediately by TransIt-mRNA reagent. The mixture was incubated at room temperature for 3 min and then aliquoted directly to cells cultured in complete media. Twelve hours after transfection when cell mRNA levels had reached a steady state, cells were washed once and changed to complete media without transfection mixture to stop uptake of transcripts and start 0-h time point. The degradation of mRNA within the cells was determined by harvesting at appropriate time points using RNeasy Mini Kit (Qiagen) and isolating total RNA according to the manufacturer’s protocol. Rat StAR message levels were determined by reverse transcription and quantitative real-time PCR as described below. The half-life for each mRNA species was calculated by linear regression fit of the time points on semi-log plots. Three independent experiments were carried out for each mRNA.
IPTG induction of luciferase chimeric constructs
The LacSwitch II system from Stratagene was employed to achieve inducible expression of luciferase. Lac repressor-controlled luciferase/StAR 3'UTR vectors were made as described previously. Y-1 cells were transfected with the Lac repressor expression vector pCMVLacI by TransIt-LT1 as described previously. Stable clones were selected by adding 150 µg/ml hygromycin B to growth media and isolate surviving colonies after 1 month. Two stably integrated clones (Y-1 LacI cells) were obtained with this method. Lac repressor-controlled luciferase vectors were then transiently transfected into Y-1 LacI cells. IPTG was added at 5 mM concentration 24 h later to induce expression of luciferase. The experiments shown in the paper were conducted with the clone that exhibits highest induction by IPTG. Separate cultures were lysed and analyzed for luciferase activity as described previously after 2–36 h.
Western blot analysis
For StAR protein blot, cells were washed once in PBS and harvested with RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 1% NP-40, 0.25% deoxychoic acid, 0.05% SDS, 40 mM NaF, 10 mM sodium molybdate, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1% pro-tease inhibitors cocktail from Sigma). Lysate was passed through a 25 gauge needle six times, centrifuged at 12 000 g speed for 10 min at 4 °C and supernatant collected. The lysates were assayed for protein concentrations using BCA protein assay kit (Pierce Bio-technology, Inc., Rockford, IL, USA). Sixty micrograms proteins were loaded on each lane, resolved on 10% SDS-PAGE gel, and electrophoretically transferred to nitrocellulose membranes. Following transfer, the membrane was incubated in blocking buffer tris buffered saline with tween (TBST) with 5% non-fat milk for 1 h, washed with TBST, incubated with an antibody raised against recombinant mouse StAR protein (Dr Dale Buck Hales, University of Illinois at Chicago, USA) for 1 h, followed by three washes with TBST and incubation with horseradish peroxidase-conjugated anti-rabbit IgG containing 1% milk and another three washes. Protein bands were visualized by ECL reagent (Amersham Biosciences) and exposing the membrane against Hyperfilm (Amersham Biosciences).
Real-time RT-PCR
StAR and luciferase mRNA levels in these assays were determined by real-time RT-PCR using specific primers that bind to ORF or untranslated regions of StAR and luciferase genes. Total cellular RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. When mRNAs produced from transfected DNA vectors were measured, an additional step of extensive DNaseI digestion was employed to remove transfected DNA vector contamination in real-time PCR. The DNaseI treatment was performed using Qiagen’s RNase-free DNase kit at 37 °C for 4 h. Effective removal of DNA vectors was confirmed by the extremely low signal of no reverse transcriptase controls in real-time PCR (<0.1% of samples with reverse transcriptase). RNA concentrations were quantitated in triplicates (<5% S.D.). Total RNA (1.5 µg) was used for cDNA synthesis by SuperscriptIII reverse transcriptase (Invitrogen) per manufacturer’s protocols. cDNA products were diluted to 100 µl, from which 3 µl were used for each well in 96-well plate real-time PCR. The primer pair 5'-TGGAAGAACAAATCCCTGGGA-3' and 5'-TGCTTTCTGTGGTAGTGCTGCA-3' was used to amplify a 5'UTR region within rat but not mouse StAR mRNAs. To detect luciferase messages, the primer pair 5'-AGTATGGGCATTTCGCAGCC-3' and 5'-CAACCCCTTTTTGGAAACGAAC-3' that bind within luciferase ORF were used. Specificity of these primers was indicated by a single sharp peak within the dissociation curves. Real-time PCR was performed on a Bio-Rad MyiQ single channel real-time PCR machine, using reagents purchased from Bio-Rad. Data were collected by linked computer and analyzed using Bio-Rad MyiQ software. All samples were done in triplicates.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The two transcripts produced from mouse or rat StAR gene share the same 5'UTR, open reading frame and a 0.7 kb region of 3'UTR. The 3.5 kb transcript contains an additional 1.9 kb extended 3'UTR (Fig. 1
). To test whether the alternatively generated 3'UTRs affect StAR expression levels, we cloned cDNAs corresponding to 1.6 and 3.5 kb rat StAR mRNA into CMV promoter driven expression vectors. We also deleted the 3'UTR entirely to make a StARdUTR vector. The control vector StAR(–) (i.e. pCINeo) did not have cDNA inserts (Fig. 2A). After transfecting these vectors into steroidogenic MA-10, Y-1, and non-steroidogenic COS-1 cells, we found marked differences in the amounts of expressed StAR proteins. As shown in Fig. 2B, in MA-10 cells basal StAR is virtually undetectable. StARdUTR, StAR1.6k, and StAR3.5k express proteins in the ratio of approximately 15:5:1 as determined with the Image Quant 5.2 software. For Y-1 cells, after subtracting high basal StAR level, the pattern is similar to MA-10 cells. In COS-1 cells, StAR3.5k produces more protein than in MA-10 or Y-1 cells. There are more precursor (p37) forms in COS-1 cells, indicative of slower processing. The amounts of StAR transcripts in transfected MA-10 cells, as assessed by real-time RT-PCR, are consistent with protein levels, ruling out effects of 3'UTR on translation (Fig. 2C).
Since the transcriptional rates of these vectors are the same, the observed differences reflect effects of StAR 3'UTR on transcript cytoplasmic stability and/or the efficiency of transfer of nascent nuclear transcripts to the ribosomes. This involves competition between formation and export of ribonucleoproteins from the nucleus versus nuclear degradation of transcripts. The level of protein from StARdUTR represents 50% of that obtained by stimulation of the endogenous gene in MA-10 cells with 1 mM 8-Br-cAMP for 3 h. Strikingly, pregnenolone formation in transfected cells did not increase significantly above low basal levels (data not shown here) despite much elevated StAR protein. Recently, we also found that transfected StAR protein is rapidly and efficiently phosphorylated upon cAMP stimulation. Nevertheless, steroidogenesis rates are not enhanced (unpublished data), suggesting factors other than StAR might be rate limiting in acute steroidogenesis. Similar results were also obtained with equivalent mouse vectors. This result does not conflict with cholesterol metabolism seen with similar vectors in COS-1 cells which are low compared with even basal MA-10 activity. This deficiency is being pursued in a separate study.
StAR 5'UTR and ORF are not involved in lowering expression
We tested whether StAR 5'UTR and ORF could negatively impact mRNA levels by transfecting MA-10 cells with parallel luciferase–StAR 3'UTR chimeric constructs (Fig. 8A). Figure 8B shows that the short 3'UTR lowers luciferase expression 2.5-fold compared with the vector without 3'UTR, while the long 3'UTR reduces it almost 20-fold. In luciferase construct transfection experiments shown here and in later figures, the activity measurements directly reflect luciferase protein levels. We also measured luciferase mRNA levels produced from these vectors by real-time RT-PCR and found them to be consistent with activity (Fig. 8C). Therefore, no translational inhibition by 3'UTR is apparent. The effects of 3'UTRs on luciferase chimeras are thus indistinguishable from those on cloned StAR vectors, including 5'UTR and ORF. We conclude that StAR 3'UTRs function the same in chimeric constructs as in native gene and that the experimental effects derive entirely from 3'UTR.
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We sought to measure the half-lives of StAR transcripts in steroidogenic cells. Earlier reports showed that StAR messages are stabilized by transcriptional inhibitors, including actinomycin D, DRB, and 
-amanitin (Clark et al. 1997, Zhao et al. 2005). This phenomenon was also observed when we used StAR or luciferase–3'UTR chimeric constructs to examine degradation of corresponding messages after transcriptional arrest (data not shown here). We therefore developed a new method to study decay kinetics of exogenous StAR transcripts. Rat StAR cDNAs with no 3'UTR, short 3'UTR (1.6 kb), or long 3'UTR (3.5 kb) were cloned into T7 promoter driven in vitro transcription vectors, followed by a 90-base synthetic poly(A) tail. After linearization, these vectors were transcribed in vitro using Ambion’s T7 mMessage mMachine system. The reaction also contains 5'RNA cap analog and capping enzyme so that mRNAs generated are capped and polyadenylated as native genes. Transcription products were checked on gel and confirmed to be of uniformly correct sizes with no signs of degradation (data not shown). These rat StAR transcripts were then delivered into MA-10 cells by a liposome-based mRNA transfection method. Initial experiments showed that the transcripts reached a steady-state level within the cell after 12 h (Fig. 9A), which was determined by the balance between cellular uptake and degradation. Further mRNA uptake was then stopped by washing away the transfection media. The decline of StAR mRNA was measured by real-time RT-PCR. Transfected rat StAR was distinguished from endogenous mouse StAR transcripts using selective primers targeting a non-conserved region in 5'UTR. No signals were seen in non-transfected cells (data not shown). Figure 9B shows results from one representative experiment. The decay of transfected StAR mRNAs followed first-order kinetics. Three independent experiments were conducted and their statistics summarized in Fig. 9C. StAR3.5kb is approximately twofold more labile than StAR–no UTR, with StAR1.6kb exhibiting intermediate stability. Their half-life values are in the range of determined rates for the decline of StAR mRNA after the removal of Br-cAMP stimulus (Zhao et al. 2005), although there the loss of 3.5 kb mRNA was about twice as rapid. Importantly, the differences between their rates of degradation are modest compared with the steady-state differences (Figs 2 and 8).
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To investigate whether these differences in steady-state levels derived from the length of the 3'UTR or specific sequence features, we made antisense constructs in which the 3'UTRs were replaced with the reverse complement of the original sequences (Fig. 3A). This removes sequence-specific elements but retains effects of length and possibly some features of secondary structures determined by complementary internal sequences. These constructs were transfected into MA-10 and Y-1 cells. Figure 3B shows that in all cell types, the antisense and sense short 3'UTR vectors produced about the same level of luciferase. By contrast, the antisense long 3'UTR expressed threefold more luciferase than sense vector. The antisense long 3'UTR vector was nevertheless expressed at three times lower levels than the antisense short 3'UTR vector. In COS-1 cells, similar trends were maintained but with smaller differences. The length of the 3'UTR alone, therefore, appears to be a factor that reduces luciferase expression levels. This will be presented in more detail in Fig. 10 where we compare the effects of several other 3'UTR modifications to dissect out the contribution of 3'UTR length to expression. The sequence-specific destabilizing effect, which manifests as differences between antisense and sense constructs, is only found with the long 3'UTR and is somewhat cell selective (Y-1>MA-10>COS-1). We additionally deleted the terminal 300 bases from the 2.5 kb 3'UTR which comprised the AURE region. This deletion had a modest 1.5-fold stabilizing effect on luciferase expression (Fig. 3C).
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These steady-state levels of mRNA are determined by the formation of cytoplasmic transcripts as well as their degradation. Although transcription from the same promoter should be the same for each vector, the formation of mature cytoplasmic transcripts additionally involves post-transcriptional nuclear processes, including splicing, polyadenylation, ribonucleoprotein packaging, and degradation which precede export to the cytoplasm for translation (Fig. 7A). In the natural gene, intron complexes that are also involved in splicing direct many of these steps, thus greatly enhancing the effectiveness of mRNA export relative to the transfected cDNA used here (Moore 2002, Vinciguerra & Stutz 2004). These post-transcriptional nuclear processes are likely to be substantially affected by the 3'UTR and may account for some of the steady-state expression differences described in the previous sections.
In order to assess the effect of extended 3'UTR on the rates of appearance of mRNA in the cytoplasm, we made IPTG-inducible luciferase–StAR 3'UTR chimeric vectors (Fig. 7B), in which the expression of luciferase gene is under control of the Lac operon. These constructs allow us to initiate transcription with IPTG and then assay the subsequent formation of luciferase as a measure of mRNA which reaches the cytoplasmic ribosome. We also generated Y-1 cells lines stably integrated with the Lac repressor gene (Y-1 LacI cells). After transfection into Y-1 LacI cells, these vectors show low basal luciferase expression. The addition of IPTG removes Lac repressor binding and induces each promoter. Since they are identical, IPTG should produce similar transcription rates for each vector. Based on our previous experiments, luciferase activity is directly proportional to mRNA levels (Fig. 8). The rate of increase for luciferase, therefore, measures the appearance of mRNA at the cytoplasmic ribosomes. This rate depends on the constant rate of transcription combined with the overall rate of nuclear processes that results in export to the cytoplasm. The steady-state level of mRNA is determined by this cytoplasmic appearance rate and the cytoplasmic degradation rate of the transcript. As shown in Fig. 7C, after IPTG addition, all three vectors show the same brief lag time (2 h) and then increase linearly for approximately 24 h at very different rates. The zero-order cytoplasmic appearance rates for each vector were established by fitting linear regressions to the luciferase activities from 2 to 24 h (Fig. 7D).
Table 1 summarizes the cytoplasmic appearance rates as determined from linear regression analyses of the increases seen in Fig. 7C after the constant 2-h lag period. These rates are compared with the mRNA degradation rates derived from the first-order plots shown in Fig. 9. The calculated ratio of these two rates should determine the steady-state levels generated by these constructs. As shown in Table 1, these ratios generally parallel the steady-state levels. This confirms that most of the differences in the steady-state luciferase mRNA derived from the effects of 3'UTR on post-transcription nuclear processes. By contrast, large differences in mRNA steady state caused solely by the differences in mRNA stability would be indicated by constant initial zero-order rates for transcription and post-transcriptional nuclear processes but different times to reach the steady states that are proportional to the degradation rates.
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The extended StAR 3'UTR comprises two distinct sequences: an AURE of about 0.3 kb and an upstream region of about 1.4 kb. We have shown that deletion of the AURE region leads to a modest increase in luciferase expression (Fig. 3C). To further examine the effect of AURE on basal stability, we made wild-type and mutant rat AURE vectors (Fig. 5A), which have UUAUUUAU motifs mutated to disrupt interaction with destabilizing AURE-binding proteins such as TIS11b (Hudson et al. 2004). Figure 5B shows that in Y-1 and MA-10 cells, StAR AURE causes 40% decline in luciferase expression. This corresponds closely to the 50% increase produced by deletion of the whole AURE region (Fig. 3C). The decline is reversed by mutation of the upstream two AURE motifs. In COS-1 cells, however, the AURE sequences have no significant effect on luciferase activity. Mutation of the third UUAUUUAU motif has no further effects in any cell types. These results confirm that the AURE confers a modest effect on basal destabilization of the extended 3'UTR, but far less than 20-fold, as shown in Figs. 2 and 8. Destabilization by AURE only functions in steroidogenic cells and can be achieved with only two UUAUUUAU motifs.
A 700-base sequence upstream of the AURE region reproduces most of the destabilizing effect of the extended 3' UTR
We also examined the effect of the region upstream of AURE (684–2115) by making corresponding luciferase chimeric constructs (Fig. 4A). The results in Fig. 4B show that in steroidogenic (Y-1 and MA-10) as well as non-steroidogenic (COS-1) cells, the upstream region substantially lowered luciferase level (vector 684–2115), while the antisense vector 684–2115as exhibited threefold higher luciferase activity. This destabilization by specific sequences in the extended 3'UTR is similar to the difference between sense and antisense long 3'UTR vectors in Fig. 3B. This suggests that the region provides a sequence-specific destabilization effect that functions in parallel with the length effect shown by the antisense vectors.
To further define basal instability determinants within the upstream region, we examined separate sequences of this region (Fig. 6A, 684–1043, 684–1403, 684–1763, and 1043–1763). As shown in Fig. 6B, 684–1043 has about the same luciferase activity as the parent vector pGL3P, indicating that this region does not contribute to destabilization. The vector 1043–1763 was almost as effective as the complete upstream region, while its antisense control had threefold higher activity. The sequence between 1043 and 1763 therefore accounts for most of the sequence-specific destabilization. We designate this sequence the basal instability region (BIR).
3'UTR length-luciferase activity plot separates non-specific length effects from sequence-specific destabilizing elements
To better dissect the AURE, BIR, and length effects, we have plotted luciferase activities of several construct against their 3'UTR lengths. As shown in Fig. 10A, for sequences that do not contain the BIR, there is a general inverse relationship between steady-state expression and 3'UTR lengths when extended beyond 300–350 bases (dotted line). This appears as a minimum length of 3'UTR for the introduction of the length effect. The correlation appears to be independent of the sequences used. For example, the vectors UTRS, UTRSas, and 1043–1763as are about the same length (700 bases) and show very similar expression levels. In these constructs, only the length effect is manifested. On the other hand, 3'UTR sequences which contain the BIR (1043–1763, 684–2115, UTRL) are displaced to much lower stabilities relative to their lengths. The shape of the plots suggests that the BIR sequence is equivalent to a non-specific sequence of three to four times the length. The effect of AURE region appears as additive with the BIR/length effects. Figure 10B summarizes the BIR and the AURE sequence elements within StAR extended 3'UTR and their different effects in steroidogenic and non-steroidogenic cells.
Introns mitigate the destabilizing effects of extended StAR 3' UTR
The impact of post-transcriptional nuclear steps raises important questions about differences in these processes between transfected cDNA vectors and the natural gene. Introns are involved in many aspects of gene expression, including transcription, polyadenylation, mRNA export, localization, decay, and translational efficiency (Le Hir et al. 2003, Nott et al. 2003; see Fig. 7A). Therefore, we cloned the mouse genomic StAR locus with all six introns and complete extended 3'UTR into the same expression vector (mStAR3.5/introns, Fig. 11A). As shown in Fig. 11B and C, although it harbors the same long 3'UTR as mStAR3.5k, mStAR3.5/introns vector generates several fold more StAR protein and mRNA than mStAR3.5k, comparable with mStAR1.6k. This suggests that StAR introns enhance StAR gene expression. This may arise by partially offsetting the destabilizing effects of long 3'UTR or by directing polyadenylation to favor the more stable 1.6 kb transcript.
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We first found that the long 3'UTR from 3.5 kb form substantially lowers StAR and luciferase protein and mRNA expression by about 20-fold. The effect of the short StAR 3' UTR was only two- to threefold (Figs 2 and 8). Since the differences are similar for either StAR or luciferase, they are due solely to effects of the extended 3'UTR. The differences are similar for mRNA and protein and therefore do not reflect the effects of 3'UTR on translation. The effects of 3'UTR on expression were also retained in the non-steroidogenic COS-1 cells although with smaller differences. Thus, the downregulation of transcripts containing the extended StAR 3'UTR is preferentially enhanced by factors in steroidogenic cell lines but is not specific to these cells.
The long 3'UTR constructs retain the internal poly(A) signal used to generate the short form. However, we have previously shown that changes in the 3.5 kb mRNA derived from the StAR3.5 vector were similar whether we used primer pairs targeted to the extended 3'UTR or to the ORF (Zhao et al. 2005). Thus, intermediate polyadenylation appears to be minimal from cDNA vectors, in contrast to the natural gene. The strong SV40 poly(A) signal, which is inserted in all these constructs may also favor terminal polyadenylation (Carswell & Alwine 1989, Legendre & Gautheret 2003). The SV40 poly(A) signal enhances the expression of these StAR vectors compared with those containing only the natural polyadenylation signals (Zhao et al. 2005). Data presented here and in the previous paper suggest that SV40 polyadenylation may be more effective for short 3'UTR.
StAR mRNA turnover in Y-1 cells is completely halted by transcriptional inhibitors (Zhao et al. 2005). We confirmed this finding by showing that the addition of actinomycin D to StAR expression vectors resulted in extremely slow mRNA degradation (half times > 10 h, data not shown). This apparent dependence of mRNA stability on transcription has been reported previously for other labile mRNA but has not been well explained (Goldberg et al. 1991, Stacey et al. 1994, Foster et al. 2001). Since this typical approach was excluded, we used direct transfection of in vitro-transcribed StAR mRNA into MA-10 cells to measure mRNA degradation rates. The first-order rates indicated that StAR mRNAs are destabilized by the extended 3'UTR (Fig. 9), but the modest effects were too small (twofold) to account for the steady-state differences (20-fold) seen with expression vectors. The degradation half-times (3.5 h for 3.5 kb and 5 h for 1.6 kb) were, however, similar to those seen in Y-1 cells after the removal of the Br-cAMP stimulus (2 and 4 h; Zhao et al. 2005).
The large effects of 3'UTR on steady-state mRNA levels occur after the shared transcription process and prior to translation. IPTG initiation of Lac-suppressed luciferase/StAR chimeras showed a 2-h delay independent of 3'UTR which precedes the induction (Fig. 7). This presumably reflects the time for LacO derepression and promoter activation, which should be similar for each of these constructs. We have established that the increase in expression of luciferase then follows zero-order rates that were greatly attenuated by the long 3'UTR. This fivefold slower cytoplasmic appearance rate coupled with the twofold faster degradation accounts for a tenfold lower steady-state level, which closely matches the 15 to 20-fold observed differences (Table 1). The maintenance of widely different zero-order rates for similar durations is also completely inconsistent with a dominant influence of cytoplasmic mRNA degradation. Instead, the reduced rates reflect decreased efficiency in nuclear processes between transcription and entry into the cytoplasm. Polyadenylation and formation of RNA-protein complexes prior to nuclear export compete with nuclear degradation (Vinciguerra & Stutz 2004). Polyadenylation clearly plays a role since we previously showed that the inclusion of the SV40 polyadenylation signal appreciably elevated the steady states (Zhao et al. 2005). Our data provide evidence that the long 3'UTR decreases the delivery of mRNA to the cytoplasm by favoring the nuclear degradation process.
We have systematically manipulated the 3'UTR of luciferase chimeras to resolve several 3'UTR sequence contributions that each selectively diminish expression of 3.5 kb StAR mRNA. A set of antisense 3'UTR constructs indicated that increased 3'UTR length produces a non-specific decrease in stability (Fig. 10), which accounts for all the effect of the short 3'UTR and some of the loss produced by the long 3'UTR. This length effect is seen in COS-1 cells although less than in Y-1 and MA-10 cells. The diminished expression difference between short and long StAR 3'UTR with endogenous polyadenylation may arise from stimulation of this length effect by the inserted SV40 signal. Sequence-specific destabilization further contributes to threefold destabilization by long 3'UTR (Fig. 3).
We have resolved two sequence-specific mechanisms. Deletion of the AURE or separate introduction into a luciferase chimera indicated that the isolated AURE caused a modest decrease in basal stability (40%) that was selective for steroidogenic cells (Fig. 5). The destabilization effect was removed by mutation of two of the AURE motifs. The adjacent upstream region surprisingly produced much greater losses in expression. A sequence of 700 bases (1043–1763) produced an appreciably large destabilizing effect on the extended 3'UTR (Fig. 6). We termed this the basal instability region (BIR). In Fig. 10, we show that destabilization by the extended 3'UTR that contains BIR exhibits a parallel length effect but with an enhanced effectiveness of about fourfold. We were concerned that the use of antisense sequences could retain RNA secondary structures. However, several different sequences of about 700 bases produce similar effects. This confirms that the length effects are relatively non-specific. Increased spacing between stop condon and poly(A) tail can diminish the formation of export-competent ribonucleoprotein complexes. This could favor nuclear exosome degradation (Colgan & Manley 1997) and account for the non-specific length effect.
The minimally functional sequences of BIR are still unknown. We used the mfold web server to examine possible RNA secondary structures of BIR (Zuker 2003). Some internal basepairings and non-paired loops were predicted, but no particular strong secondary structures were identified. Furthermore, antisense BIR sequences, which should retain most of the secondary structural features of BIR, show threefold higher luciferase expression level (Fig. 6B). This argues against a role of secondary structure in the destabilizing effect of BIR. We also aligned the rat BIR sequences with the corresponding mouse StAR 3'UTR, and found a region of about 300 bases that is over 80% identical, with three other shorter regions (25–100 bases) also showing > 80% identity. Whether these sequences harbor cis-elements that mediate BIR’s destabilizing effect awaits future examination. One possible hypothesis for the mechanism of BIR is that this region may contain targets for endogenous miRNA. Indeed, our search of BIR against the Sanger Institute’s miRNA database returned one mature miRNA (mmu-miR-706) with only one base mismatch, although the miRNA is from a murine database. It is possible that more complete miRNA databases in the future will help identify potential targets within BiR.
We hypothesize that destabilization by BIR is enhanced in steroidogenic cells, rendering the 3.5 kb transcript more labile under basal conditions and primed for fast stabilization responses to external stimuli. The AURE contributes only modestly to these basal stability mechanisms. Recent studies using bovine adrenocortical cells indicate that AURE in VEGF responds to stabilization by cAMP activation of HuR and destabilization by TIS11b. These proteins compete for AURE sequences (Cherradi et al. 2006). The AURE in the StAR 3'UTR match the consensus binding site for TIS11b homodimers (Ciais et al. 2004). Another AURE-binding protein AUF1 has also been implicated in regulation of mRNA stability (Raineri et al. 2004). We have recently found that each of these proteins is highly expressed in MA-10 and Y-1 cells (unpublished data).
In higher eukaryotes, introns have been reported to affect many stages of gene expression, including transcription, polyadenylation, mRNA export, translation, and mRNA turnover (Le Hir et al. 2001, Furger et al. 2002, Proudfoot et al. 2002, Wilkinson & Shyu 2002). The magnitude of intron effects varies among genes and is dependent on their locations (Nott et al. 2003). The low expression derived from the 3.5 kb cDNA vector prompted us to ask whether the endogenous StAR gene had mechanisms to enhance transcript formation. Our results with a cloned minigene clearly show that introns enhance StAR expression, partially counteracting the destabilization of the extended 3'UTR (Fig. 11). However, since we have not identified the transcripts we cannot exclude the possibility that introns affect polyadenylation to produce the more stabile 1.6 kb transcript.
Artificially long mRNA half-lives measured by transcriptional inhibitors have previously been reported. This may be a more wide spread problem than often noticed (Goldberg et al. 1991, Stacey et al. 1994, Foster et al. 2001). We have developed methods which accurately reflect the rapid responses seen in steroidogenic cells and have shown that the 3.5 kb message is indeed less stable basally than the 1.6 kb message (Fig. 9). Alternative polyadenylation is a common yet poorly studied phenomenon (Edwalds-Gilbert et al. 1997, Beaudoing et al. 2000). In some instances, it has been established that messages generated by this mechanism harbor distinct 3'UTR sequence elements with important regulatory implications (Touriol et al. 1999, Caballero et al. 2004, Hall-Pogar et al. 2005). Our results suggest the same applies to rodent StAR gene. Based on all the information, we hypothesize that the 3.5 kb message serves as a fast responder to external stimuli due to its intrinsically short half-life conferred by BIR and potential regulation through AURE, while the 1.6 kb message reacts more slowly to establish a baseline expression level. The combination of these two responses makes the StAR gene adaptable to a variety of physiological conditions and steroidogenic needs.
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Received 13 September 2006
Accepted 9 October 2006
Made available online as an Accepted Preprint 14 November 2006
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P < 0.05 compared with 1.6 kb by Student’s t-test.
