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Posttranscriptional Destabilization of the Bradykinin B1 Receptor Messenger RNA: Cloning and Functional Characterization of the 3′-Untranslated Region
Molecular Cell Biology Research Communications 1, 29 –35 (1999) Article ID mcbr.1999.0105, available online at http://www.idealibrary.com on
Posttranscriptional Destabilization of the Bradykinin B1 Receptor Messenger RNA: Cloning and Functional Characterization of the 39-Untranslated Region Xiaofeng Zhou, 1 Gregory N. Prado, Minhua Chai, Xionghu Yang, Linda Taylor, and Peter Polgar Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118
Received January 22, 1999
have been characterized (2, 4 – 6). The BKB1 receptor is only minimally expressed under normal physiological conditions. However, functional BKB1 receptor number is rapidly induced under many inflammatory conditions such as arthritis, cystitis, UV irradiation, colitis and hyperalgesia, and inflammation (3, 7–11). Our previous study illustrated that the expression of this gene is regulated not only by transcriptional activation but also by post-transcriptional mRNA stabilization (12). The 39-untranslated region (39-UTR) of the mRNA has proven to be a primary site for the regulation of mRNA stability in eukaryotic cells (13, 14). Recent evidence also suggests that the regulation of the mRNA stability involves the binding of trans-factors to the cis-element located in the 39-UTR (15, 16). However, other regions of the mRNA, such as the coding region, may also be involved in the regulation of mRNA stability (17). Human BKB1R cDNA as well as the genomic DNA have been cloned and characterized (6, 18). The 39-UTR of BKB1R has not been defined yet. As part of an effort to learn more about the role of 39-UTR of BKB1R mRNA, we cloned and characterized this region.
We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3*-untranslated region (3*-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFa, phorbol ester, bradykinin, and desArg 10-kallidin contain the same 3*-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3*-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3*-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression. © 1999 Academic Press
EXPERIMENTAL PROCEDURES Cloning and sequencing. A 39-RACE procedure was used to clone the 39-UTR according to the protocol provided by manufacturer (Life Technologies) with minor modification. Briefly, first stand cDNA is synthesized with SuperScriptII reverse transcriptase using the oligo dT adapter (Life Technologies) as primer and 3 mg of total RNA isolated from IL-1b treated IMR 90 cells as template. The reverse transcription (RT) products are treated with 2 unit of E. coli RNase H. The RT products are amplified by PCR with 59-GCTCC AATAT CTTCA TCCCA TAGG-39 (corresponds to 13255 to 13278 of BKB1R genomic sequence, Ref. 6, GenBank accession number U48827 and U22346) as sense primer and amplification primer provided by manufacturer as anti-sense primer. The product is then sub-
Bradykinin (BK) receptors are members of the seven transmembrane, G-protein coupled receptor family (1). A large body of evidence points to the involvement of BK receptors in inflammation and shock leading to hypotension and pain (2, 3). Two subtypes of bradykinin receptor (BKB1 and BKB2) with variable ligand binding affinity and different agonists and antagonists Sequence data from this article have been submitted to GenBank under Accession No. AF117819. 1 To whom correspondence should be addressed. Fax: 617-6385339. E-mail: [email protected]. 29
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cloned into PCRII vector and sequenced using a plasmid double-stranded DNA sequencing kit USD (Cleveland, OH) and an ABI automatic sequencer Model 373A.
blunted BamHI sites of pGL 3-P. The chimeric constructs were verified by restriction analysis and partial DNA sequence analysis. Cell culture and transfections. IMR90-SV40 (IMRSV) and IMR90 cells were grown in minimal essential medium with 10% fetal bovine serum. Rat–1 cells were grown in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum. IMRSV cells were transiently transfected using DEAE– dextran method (20). IMR 90 and Rat 1 cells were transfected using the DNAcalcium phosphate precipitation method (21). To minimize the possible effects of different transfection efficiencies, the transfected cells were pooled 36 h after transfection and re-seeded into separated plates.
Reverse transcription (RT) assay. RT reaction is carried out with rTth reverse transcriptase (Perkin– Elmer, Branchburg, NJ) using 3 mg of total RNA as template. The primers used are primer 1, 59-GTCAC AAAGA ATTGA TAAGA AAGCC AAGC-39, corresponding to the region that is present 39 of the poly-A site in the genomic sequence and primer 2, 59-CAATG CTGTT TTAAT TCCGC CAG-39, corresponding to the 59 of the poly-A site. The RT products are amplified by PCR with primer 3, 59-CTGGC TGCGA TCGTC TTCTT CAAC-39, located in the coding region 400 bp upstream of the stop codon, as sense primer and the primers for RT reaction as antisense primers (see Fig. 3, upper panel). As control, RT-PCRs were also performed with a template containing BKB1R genomic DNA.
Luciferase assay. Extracts of transfected cells were prepared 48 h after transfection. The cell debris was removed by centrifugation at 10,000g for 1 min. Luciferase activity was determined using 10 ml clarified extracts and 100 ml of luciferase assay substrate (Promega) with a dual luciferase system following the procedure provided by manufacturer (Promega, Madison, WI).
Northern blot analysis. Total RNA was extracted from cells by the guanidinium thiocyanate-phenol– chloroform procedure according to the methods described previously (19). RNA was quantitated by ultraviolet absorbance at 260 nm, and 10 mg denatured RNA was electrophoresed on a 1% agarose/formaldehyde denaturing gel and transferred to nylon membranes (DuPont–NEN). The blots were hybridized sequentially with 32P-labeled cDNA probes, at 65°C in a Rapid-hyb buffer (Amersham Life Science, Amersham, UK) and washed as described (19). All incubations for Northern blots were done in duplicate. The cDNA probe for BKB1R was produced as described previously (12). The cDNA probe for luciferase was released from pGL 3-Promoter vector (Promega, Madison, WI) by digestion with NcoI and XbaI (New England Biolabs, Beverly, MA).
Determination of the mRNA half-life. To determine the half-life of luciferase mRNA, actinomycin D (5 mg/ ml) was added to cells 48 h after transfection. RNA was isolated from samples of cells taken at several time points after the addition of actinomycin D. Northern blot analysis was performed as described above with a cDNA probe for the luciferase gene. RESULTS The 39-RACE procedure was performed to clone the BKB1R gene 39-UTR. The product was PCR amplified and showed 2 bands on the gel (Fig. 1A). The first band was located at approximately 200 bp and the second at 700 bp (lanes 2 and 3). As control, the 39-RACE product was also amplified with only sense or anti-sense primers and run on the gel (lanes 1 and 4 respectively). The sense primer gave no product. The anti-sense primer (amplification primer provided by Life technologies) alone, gave one band at the 700 bp, the same position as for the second band. This suggested that the 700-bp band was a nonspecific product. Both the 200- and 700-bp product were sequenced. The sequence of the 200-bp product contained the last 57 bp of the BKB1R coding region followed by 14 bp of 39-UTR and 110 bp of poly-A tail (Fig. 1B). The 700-bp product proved 98% identical to the human mitochondria DNA sequence from 2317 to 2637 (GenBank Accession No. D38112) which confirmed that this was a non-specific product. We compared this 200-bp product with the BKB1R genomic sequence (Fig. 1B). The most striking feature of this 39-UTR is that it is only 14 bases long. To our knowledge it is the shortest 39-UTR reported thus far.
Constructs. The plasmid vector pGL 3-Promoter (pGL 3-P, Promega) was used as the backbone to generate chimeric constructs. The structure of the constructs is shown in Fig. 6. The pGLtailless was generated by digestion of pGL 3-P with XbaI and BamHI to remove a 262-bp fragment and then followed by blunting and ligation. The pGL-B1gen was generated by inserting a BKB1R genomic sequence of 13330 to 13891 bp into the XbaI site of the pGL 3-P in the sense direction. The pGL-B1-39 was generated as follows: Oligonucleotides corresponding to the BKB1R 39-UTR, sense 59-CTAGA ATTAA AACAG CATTG AACCA AGAAG CTTGG CTTTC TTATC AATTC TTTGT GACAT AATAA A-39 and anti-sense 59-TTTAT TATGT CACAA AGAAT TGATA AGAAA GCCAA GCTTC TTGGT TCAAT GCTGT TTTAA TT-39 were synthesized. (Life Technologies, Inc., Gaithersburg, MD.) The sense and anti-sense oligonucleotides were annealed at equal molar ratio and then inserted into the XbaI and 30
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FIG. 1. Cloning of the Bradykinin B1 mRNA 39-UTR. (A) The first stand cDNA is synthesized as described in the experimental procedure and amplified by PCR with a 59-primer indicated in Fig. 3 as sense primer and amplification primer provided by manufacturer as antisense primer (lanes 2 and 3). As a control, the RT products are also amplified with 59-primer alone (lane 1) or amplification primer alone (lane 4) The PCR products were run on a 2% agarose gel along with the molecular weight markers. (B) Sequence of the cloned product is compared with the genomic sequence reported by Yang et al. (6). Sequence motifs discussed in the text are underlined.
As illustrated in Fig. 2, an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon was used. The polyadenylation site, also called cleav-
age site, was between CA bases. Sheets et al. (22) reported that 59% of polyadenylation signals they surveyed contained a C at position 21 and 71% contained
FIG. 2. Comparison of the elements of the polyadenylation signal of Bradykinin B1 receptor mRNA with the optimum mammalian polyadenylation signal. Sequence elements of the bradykinin B1 receptor mRNA polyadenylation signal is compared with the optimum mammalian polyadenylation signal modified from Chen et al. (23). The length of the elements and the distance between these elements (in nucleotides) are shown by the numbers on the bottom. 31
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These UREs have been shown to be functionally important for the polyadenylation process (24). To find out whether this is the only species of the 39-UTR of the BKB1 mRNA, a reverse transcription (RT) assay was performed with the primers corresponding to the regions that present at either 39 (Primer 1) or 59 (Primer 2) of the poly-A site. The RT products were then amplified with primer 3 which was located in the coding region 400 bp upstream of the stop codon as sense primer and the primers for RT reaction as antisense primers (see Fig. 3, upper panel). As control, PCRs were performed with a template containing BKB1R genomic DNA and primers 2 and 3 or primers 1 and 3 (lanes 2 and 4). RT reaction with primer 2 gives a specific band at around 450 bp (lane 1), the same location as the band for control PCR with primers 2 and 3 (lane 2). RT reaction with primer 1 does not produce any visible band (lane 3). The control PCR with primers 2 and 3 shows a band at around 500 bp (lane 4). This confirmed that it is the only species of the BKB1R mRNA. To further confirm that this is the only species of the BKB1R mRNA, Northern blots were performed on RNA isolated from IMR90 cells treated with or without 100 pg/ml IL-1. For both the IL-1 treated and untreated cells, a single band appeared at approximately 1.4 KB (Fig. 4A). This is in agreement with the calculated mRNA length (Fig. 4B). As we show in Fig. 5A, various effectors, such as TNFa, phorbol ester, bradykinin, and desArg 10kallidin, increase BKB1R mRNA. To test if these effectors induced BKB1R mRNA also contain the same 39UTR, we performed RT-PCR with the same primers as used in experiments shown in Fig. 3. As show in Fig. 5B, RT-PCR with primer 2 in all cases produced a band
FIG. 3. Reverse transcription of the BKB1R mRNA. Upper panel: Illustration of the location of the primers used in the RT reaction. Lower panel: RT reaction was carried out as described in the experimental procedure. For lane 1, RT product with primer 2 is PCR amplified with Primers 2 and 3. As control, PCR was performed with a template containing BKB1R genomic DNA and primers 2 and 3 (lane 2). For lane 3, the RT product with primer 1 is PCR amplified with primers 1 and 3. The product of control PCR with BKB1R genomic DNA and primers 1 and 3 was run on lane 4. Data represent three separate experiments.
an A at position 11 suggesting that this CA dinucleotide may be an additional recognition element for the cleavage machinery. Two closely positioned downstream U-rich elements (URE) which are often located 39 of the polyadenylation site (23) were found at 7 base and 18 base downstream of the polyadenylation site.
FIG. 4. Determine the size of the BKB1R mRNA. (A) Northern blot was performed on RNA isolated from IMR 90 cells that treated with 100 pg/ml IL-1b or vehicle for 2 h. The positions of 28 S and 18 S ribosomal RNA and the RNA size marker are indicated. (B) A schematic presentation of the BKB1R mRNA. The numbers represent the number of the bases in each section of the mRNA. The information for the 59-UTR and the coding region is obtained from our previous study (6). 32
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pGL-P and pGL-B1-39. As shown in Fig. 7B, the 39UTR of BKB1R caused the similar decrease of the luciferase activity. The luciferase activities of pGLB1-39 was 15, 24, and 8% of the wild-type pGL-P in IMRSV, IMR and Rat 1 cells, respectively. To test whether the 39-UTR of the BKB1R mRNA has a role in the regulation of the mRNA stability, we transiently transfected the pGL-P and pGL-B1-39 in the IMRSV cells. After addition of actinomycin D, RNA was isolated from the cells at several time points, and northern blot was performed as described. As showed in Fig. 8B, the mRNA for wild type luciferase pGL-P proved very stabile with a half-life of about 6h. However, the mRNA for pGL-B1-39 decayed rapidly with a half-life of approximately 1 h. DISCUSSION Most eukaryotic mRNAs have a polyadenylated (poly-A) tail at the 39 end. The addition of this tail process involves both cleavage of the primary transcript at the 39 end of the mRNA and a coupled poly-
FIG. 5. Effect of various effectors on BKB1R mRNA. (A) Northern blot was performed on RNA isolated from IMR 90 cells that were treated with 2 ng/ml TNFa, 100 nM TPA, 50 ng/ml bradykinin, 100 ng/ml desArg 10-kallidin or vehicle for 2 h. The formaldehyde gel for 18 S is shown to demonstrate equal loading. (B) RT reaction was carried out as described in the legend to Fig. 3. The primers used for each reaction are indicated. (For position of each primer, see Fig. 3, upper panel.) The RT products were amplified with primer 3 and the primer for RT reaction then run on the agarose gel.
at 500 bp but no band was produced with primer 1. This suggested that the polyadenylation site of this mRNA is not affected by these effectors. To assess the involvement of the BKB1R 39-UTR in the regulation of the mRNA stability, we created a pGL-B1-39 construct by replacing the 39-UTR of the pGL 3-P luciferase with the BKB1R 39-UTR as illustrated in Fig. 6. As controls, we also generated the pGLtailless and pGL-B1gen constructs. These constructs were transfected into the IMRSV cells. Following the transfection both Northern blot and luciferase activity assays were performed. As shown in Fig. 7A and Fig. 8A, transfection of the wild type luciferase gene (pGL3-P) produced a single band on Northern blot and high luciferase activity. The pGL-B1gen, in which a 500-bp genomic sequence of the BKB1R after the 39-UTR has been inserted into the luciferase 39-UTR, also produced a high luciferase activity. The pGLtailless, which lacks the polyadenylation signal, produced multiple bands on Northern blot and a very low luciferase activity, around 4% of the wild type. The pGLB1-39 produced a single band on Northern blot and the luciferase activity is 15% of the wild-type luciferase. This suggested that the BKB1R polyadenylation signal is functional. We also tested this 39-UTR effect on the luciferase gene in different cell line. We transiently transfected the IMRSV, IMR and Rat 1 cells with
FIG. 6. The structure of the constructs used in the study. The plasmid pGL3-P was used as the backbone to generate chimeric constructs. The essential elements of the pGL3-P are indicated. The chimeric constructs were generated as described under Experimental Procedures. The open bars represent the pGL3-P sequence elements. The closed bars represent the sequence elements of BKB1R. The numbers above the closed bars represents the location of the sequence in the BKB1R gene. The triangles represent the poly-A signal. 33
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FIG. 7. Effect of BKB1R 39-UTR on Luciferase activity. (A) IMRSV cells were transiently transfected with pGL, pGL-B1gen, pGLtailless, and pGL-B1-39. Extracts of transfected cells along with untransfected cells were prepared 48 h after transfection, and the luciferase activities were assayed with a dual luciferase system (Promega). (B) IMRSV, IMR 90 and Rat 1 cells were transiently transfected with pGL and pGL-B1-39. Extracts of transfected cells were prepared 48 h after transfection, and luciferase activities were assayed. The closed bars and the open bars represent the luciferase activity of cells transfected with pGL and pGL-B1-39 respectively. The error bars represent standard deviations of three experiments.
adenylation reaction (25). A highly conserved AAUAAA sequence located 10 to 30 nucleotides 59 of the poly-A site signals for the cleavage and poly-A reaction (26a, 26b). However, we found that an alternative AUUAAA motif which overlaps with the stop codon serves as the poly-A signal in BKB1R mRNA. This alternative motif may play an important role in the expression of this rapidly induced gene.
Posttranscriptional mechanisms are critical in regulating the expression of many rapidly induced, inflammation related genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) (13, 27, 28). We previously showed that the expression of BKB1R gene is regulated in part by mRNA stability (12). It is well accepted that the 39-UTR is the primary target for the regulation of mRNA stability (13, 14).
FIG. 8. Effect of BKB1R 39-UTR on mRNA stability of Luciferase gene. (A) The RNA were isolated from IMRSV cells that were transiently transfected with pGL, pGLtailless and pGL-B1-39 for 48 h. The Northern blot was performed with a cDNA probe for luciferase gene. The positions for 28 S and 18 S are indicated. (B) IMR90 SV40 cells were transiently transfected with pGL and pGL-B1-39. After 48 h, Actinomycin D (5 mg/ml) was added to the cultures and RNA was then isolated at 0, 1, and 3 h from pGL-B1-39 transfected cells and at 0, 3, and 6 h from pGL transfected cells. Northern blot was performed as described. The formaldehyde gel for ribosomal 28S is shown to demonstrate even loading. The Northern blots shown represent one of three separate experiments. 34
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MOLECULAR CELL BIOLOGY RESEARCH COMMUNICATIONS 8. Donaldson, L. F., Hanley, M. R., and Villablanca, A. C. (1997) Trends Pharmacol. Sci. 18, 171–181. 9. Davis, C. L., Naeem, S., Phagoo, S. B., Campbell, E. A., Urban, L., and Burgess, G. M. (1996) Br. J. Pharmacol. 118, 1469 –1476. 10. Rupniak, N. M., Boyce, S., Webb, J. K., Williams, A. R., Carlson, E. J., Hill, R. G., Borkowski, J. A., and Hess, J. F. (1997) Pain 71, 89 –97. 11. Zuzack, J. S., Burkard, M. R., Cuadrado, D. K., Greer, R. A., Selig, W. M., and Whalley, E. T. (1995) J. Pharmacol. Exp. Ther. 277, 1337–1343. 12. Zhou, X., Polgar, P., and Taylor, L. (1998) Biochem. J. 330, 361–366. 13. Ross, J. (1995) Microbiol. Rev. 59, 423– 450. 14. Ross, J. (1996) Trends Genet. 12, 171–175. 15. Agellon, L. B., and Cheema, S. K. (1997) Biochem. J. 328, 393– 399. 16. Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261–14268. 17. Shetty, S., Kumar, A., and Idell, S. (1997) Mol. Cell. Biol. 17, 1075–1083. 18. Menke, J. G., Borkowski, J. A., Bierilo, K. K., MacNei, T., Derrick, A. W., Schneck, K. A., Ransom, R. W., Strader, C. D., Linemeyer, D. L., and Hess, J. K. (1994) J. Biol. Chem. 269, 21583–21586. 19. Taylor, L., Ricupero, D., Jean, J., Jackson, B. A., Navarro, J., and Polgar, P. (1992) Biochem. Biophys. Res. Commun. 188, 786 – 793. 20. Conklin, B. R., Herzmark, P., Ishida, S., Voyno-Yasenetskaya, T. A., Sun, Y., and Bourne, H. R. (1996) Mol. Pharm. 50, 885– 890. 21. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044 –1051. 22. Sheets, M. D., Ogg, S. C., and Wickens, M. P. (1990) Nucleic Acids Res. 18, 5799 –5805. 23. Chen, F., MacDonald, C. C., and Wilusz, J. (1995) Nucleic Acids Res. 23, 2614 –2620. 24. Chou, Z.-F., Chen, F., and Wilusz, J. (1994) Nucleic Acids Res. 22, 2525–2531. 25. Wickens, M. (1990) Trends Biochem. Sci. 15, 277–281. 26a.Proudfoot, N. (1991) Cell 64, 671– 674. 26b.Molecular Biology of the Cell (1994) 3rd ed., Chap. 8, pp. 368 – 371, Garland Publishing. 27. Ristimaki, A., Garfinkel, S., Wessendorf, J., Maciag, T., and Hla, T. (1994) J. Biol. Chem. 269, 11769 –11775. 28. Hattori, Y., and Gross, S. S. (1995) Biochem. Mol. Biol. Int. 37, 439 – 445. 29. Chen, C., and Shyu, A. (1995) Trends Biochem. Sci. 20, 465– 470. 30. Myer, V. E., Fan, X. C., and Steitz, J. A. (1997) EMBO J. 16, 2130 –2139. 31. Ma, W. J., Chung, S., and Furneaux, H. M. (1997) Nucleic Acids Res. 25, 3564 –3569. 32. Ma, W., Cheng, S., Campbell, C., Wright, A., and Furneaux, H. M. (1996) J. Biol. Chem. 271, 8144 – 8151. 33. Chu, W., Presky, D. H., Swerlick, R. A., and Burns, D. K. (1994) J. Immunol. 153, 4179 – 4189. 34. Newton, R., Seybold, J., Liu, S. F., and Barnes, P. (1997) Biochem. Biophys. Res. Commun. 234, 85– 89.
Several destabilization elements, such as the AUUUA motif and stem loop structure, have been characterized in the 39-UTR (13, 29). Recently, a 36-kD RNA binding protein, HuR, which binds to the AUUUA motif with high affinity and selectivity, has been identified and cloned (30 –32). An additional mechanism for regulating the RNA stability has been proposed which involves differential polyadenylation induced by extracellular stimulation and also the production of different isoforms of 39-UTR with different effects on mRNA stability (26a, 26b, 33, 34). In this study, we show that the BKB1R 39-UTR contributes significantly to the regulation of mRNA stability. The 39-UTR which we cloned for the BKB1R mRNA proved to be only 14 bases long and does not appear to contain any known destabilization elements. However, this short 39-UTR itself may serve as a regulatory element for mRNA stability. We also show that various stimulants, such as IL-1, TNFa, phorbol ester, bradykinin and desArg 10kallidin which cause substantial increases in steady state BKB1R mRNA levels do not cause differential polyadenylation in BKB1R mRNA. Alternatively, some other regions may also be involved in the regulation of mRNA stability perhaps through direct interaction with the 39-UTR or in conjunction with some trans elements. In fact, it has been shown that the stability of urokinase-type plasminogen activator receptor mRNA is regulated in part by an element located in the coding region (17). Interestingly, two AUUUA motifs are found in the coding region of BKB1R (6, 18). It has yet to be determined if these motifs are also responsible for the regulation of BKB1 R mRNA stability. ACKNOWLEDGMENTS We thank Mr. Ned Rich at the CABR DNA/Protein Analysis Core for the expert help with sequencing and Mr. Min Zhou for the help with the 39-RACE procedure. This work was supported in part by NIH Grant HL 25776.
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Posttranscriptional Destabilization of the Bradykinin B1 Receptor Messenger RNA: Cloning and Functional Characterization of the 39-Untranslated Region Xiaofeng Zhou, 1 Gregory N. Prado, Minhua Chai, Xionghu Yang, Linda Taylor, and Peter Polgar Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118
Received January 22, 1999
have been characterized (2, 4 – 6). The BKB1 receptor is only minimally expressed under normal physiological conditions. However, functional BKB1 receptor number is rapidly induced under many inflammatory conditions such as arthritis, cystitis, UV irradiation, colitis and hyperalgesia, and inflammation (3, 7–11). Our previous study illustrated that the expression of this gene is regulated not only by transcriptional activation but also by post-transcriptional mRNA stabilization (12). The 39-untranslated region (39-UTR) of the mRNA has proven to be a primary site for the regulation of mRNA stability in eukaryotic cells (13, 14). Recent evidence also suggests that the regulation of the mRNA stability involves the binding of trans-factors to the cis-element located in the 39-UTR (15, 16). However, other regions of the mRNA, such as the coding region, may also be involved in the regulation of mRNA stability (17). Human BKB1R cDNA as well as the genomic DNA have been cloned and characterized (6, 18). The 39-UTR of BKB1R has not been defined yet. As part of an effort to learn more about the role of 39-UTR of BKB1R mRNA, we cloned and characterized this region.
We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3*-untranslated region (3*-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFa, phorbol ester, bradykinin, and desArg 10-kallidin contain the same 3*-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3*-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3*-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression. © 1999 Academic Press
EXPERIMENTAL PROCEDURES Cloning and sequencing. A 39-RACE procedure was used to clone the 39-UTR according to the protocol provided by manufacturer (Life Technologies) with minor modification. Briefly, first stand cDNA is synthesized with SuperScriptII reverse transcriptase using the oligo dT adapter (Life Technologies) as primer and 3 mg of total RNA isolated from IL-1b treated IMR 90 cells as template. The reverse transcription (RT) products are treated with 2 unit of E. coli RNase H. The RT products are amplified by PCR with 59-GCTCC AATAT CTTCA TCCCA TAGG-39 (corresponds to 13255 to 13278 of BKB1R genomic sequence, Ref. 6, GenBank accession number U48827 and U22346) as sense primer and amplification primer provided by manufacturer as anti-sense primer. The product is then sub-
Bradykinin (BK) receptors are members of the seven transmembrane, G-protein coupled receptor family (1). A large body of evidence points to the involvement of BK receptors in inflammation and shock leading to hypotension and pain (2, 3). Two subtypes of bradykinin receptor (BKB1 and BKB2) with variable ligand binding affinity and different agonists and antagonists Sequence data from this article have been submitted to GenBank under Accession No. AF117819. 1 To whom correspondence should be addressed. Fax: 617-6385339. E-mail: [email protected]. 29
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cloned into PCRII vector and sequenced using a plasmid double-stranded DNA sequencing kit USD (Cleveland, OH) and an ABI automatic sequencer Model 373A.
blunted BamHI sites of pGL 3-P. The chimeric constructs were verified by restriction analysis and partial DNA sequence analysis. Cell culture and transfections. IMR90-SV40 (IMRSV) and IMR90 cells were grown in minimal essential medium with 10% fetal bovine serum. Rat–1 cells were grown in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum. IMRSV cells were transiently transfected using DEAE– dextran method (20). IMR 90 and Rat 1 cells were transfected using the DNAcalcium phosphate precipitation method (21). To minimize the possible effects of different transfection efficiencies, the transfected cells were pooled 36 h after transfection and re-seeded into separated plates.
Reverse transcription (RT) assay. RT reaction is carried out with rTth reverse transcriptase (Perkin– Elmer, Branchburg, NJ) using 3 mg of total RNA as template. The primers used are primer 1, 59-GTCAC AAAGA ATTGA TAAGA AAGCC AAGC-39, corresponding to the region that is present 39 of the poly-A site in the genomic sequence and primer 2, 59-CAATG CTGTT TTAAT TCCGC CAG-39, corresponding to the 59 of the poly-A site. The RT products are amplified by PCR with primer 3, 59-CTGGC TGCGA TCGTC TTCTT CAAC-39, located in the coding region 400 bp upstream of the stop codon, as sense primer and the primers for RT reaction as antisense primers (see Fig. 3, upper panel). As control, RT-PCRs were also performed with a template containing BKB1R genomic DNA.
Luciferase assay. Extracts of transfected cells were prepared 48 h after transfection. The cell debris was removed by centrifugation at 10,000g for 1 min. Luciferase activity was determined using 10 ml clarified extracts and 100 ml of luciferase assay substrate (Promega) with a dual luciferase system following the procedure provided by manufacturer (Promega, Madison, WI).
Northern blot analysis. Total RNA was extracted from cells by the guanidinium thiocyanate-phenol– chloroform procedure according to the methods described previously (19). RNA was quantitated by ultraviolet absorbance at 260 nm, and 10 mg denatured RNA was electrophoresed on a 1% agarose/formaldehyde denaturing gel and transferred to nylon membranes (DuPont–NEN). The blots were hybridized sequentially with 32P-labeled cDNA probes, at 65°C in a Rapid-hyb buffer (Amersham Life Science, Amersham, UK) and washed as described (19). All incubations for Northern blots were done in duplicate. The cDNA probe for BKB1R was produced as described previously (12). The cDNA probe for luciferase was released from pGL 3-Promoter vector (Promega, Madison, WI) by digestion with NcoI and XbaI (New England Biolabs, Beverly, MA).
Determination of the mRNA half-life. To determine the half-life of luciferase mRNA, actinomycin D (5 mg/ ml) was added to cells 48 h after transfection. RNA was isolated from samples of cells taken at several time points after the addition of actinomycin D. Northern blot analysis was performed as described above with a cDNA probe for the luciferase gene. RESULTS The 39-RACE procedure was performed to clone the BKB1R gene 39-UTR. The product was PCR amplified and showed 2 bands on the gel (Fig. 1A). The first band was located at approximately 200 bp and the second at 700 bp (lanes 2 and 3). As control, the 39-RACE product was also amplified with only sense or anti-sense primers and run on the gel (lanes 1 and 4 respectively). The sense primer gave no product. The anti-sense primer (amplification primer provided by Life technologies) alone, gave one band at the 700 bp, the same position as for the second band. This suggested that the 700-bp band was a nonspecific product. Both the 200- and 700-bp product were sequenced. The sequence of the 200-bp product contained the last 57 bp of the BKB1R coding region followed by 14 bp of 39-UTR and 110 bp of poly-A tail (Fig. 1B). The 700-bp product proved 98% identical to the human mitochondria DNA sequence from 2317 to 2637 (GenBank Accession No. D38112) which confirmed that this was a non-specific product. We compared this 200-bp product with the BKB1R genomic sequence (Fig. 1B). The most striking feature of this 39-UTR is that it is only 14 bases long. To our knowledge it is the shortest 39-UTR reported thus far.
Constructs. The plasmid vector pGL 3-Promoter (pGL 3-P, Promega) was used as the backbone to generate chimeric constructs. The structure of the constructs is shown in Fig. 6. The pGLtailless was generated by digestion of pGL 3-P with XbaI and BamHI to remove a 262-bp fragment and then followed by blunting and ligation. The pGL-B1gen was generated by inserting a BKB1R genomic sequence of 13330 to 13891 bp into the XbaI site of the pGL 3-P in the sense direction. The pGL-B1-39 was generated as follows: Oligonucleotides corresponding to the BKB1R 39-UTR, sense 59-CTAGA ATTAA AACAG CATTG AACCA AGAAG CTTGG CTTTC TTATC AATTC TTTGT GACAT AATAA A-39 and anti-sense 59-TTTAT TATGT CACAA AGAAT TGATA AGAAA GCCAA GCTTC TTGGT TCAAT GCTGT TTTAA TT-39 were synthesized. (Life Technologies, Inc., Gaithersburg, MD.) The sense and anti-sense oligonucleotides were annealed at equal molar ratio and then inserted into the XbaI and 30
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FIG. 1. Cloning of the Bradykinin B1 mRNA 39-UTR. (A) The first stand cDNA is synthesized as described in the experimental procedure and amplified by PCR with a 59-primer indicated in Fig. 3 as sense primer and amplification primer provided by manufacturer as antisense primer (lanes 2 and 3). As a control, the RT products are also amplified with 59-primer alone (lane 1) or amplification primer alone (lane 4) The PCR products were run on a 2% agarose gel along with the molecular weight markers. (B) Sequence of the cloned product is compared with the genomic sequence reported by Yang et al. (6). Sequence motifs discussed in the text are underlined.
As illustrated in Fig. 2, an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon was used. The polyadenylation site, also called cleav-
age site, was between CA bases. Sheets et al. (22) reported that 59% of polyadenylation signals they surveyed contained a C at position 21 and 71% contained
FIG. 2. Comparison of the elements of the polyadenylation signal of Bradykinin B1 receptor mRNA with the optimum mammalian polyadenylation signal. Sequence elements of the bradykinin B1 receptor mRNA polyadenylation signal is compared with the optimum mammalian polyadenylation signal modified from Chen et al. (23). The length of the elements and the distance between these elements (in nucleotides) are shown by the numbers on the bottom. 31
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These UREs have been shown to be functionally important for the polyadenylation process (24). To find out whether this is the only species of the 39-UTR of the BKB1 mRNA, a reverse transcription (RT) assay was performed with the primers corresponding to the regions that present at either 39 (Primer 1) or 59 (Primer 2) of the poly-A site. The RT products were then amplified with primer 3 which was located in the coding region 400 bp upstream of the stop codon as sense primer and the primers for RT reaction as antisense primers (see Fig. 3, upper panel). As control, PCRs were performed with a template containing BKB1R genomic DNA and primers 2 and 3 or primers 1 and 3 (lanes 2 and 4). RT reaction with primer 2 gives a specific band at around 450 bp (lane 1), the same location as the band for control PCR with primers 2 and 3 (lane 2). RT reaction with primer 1 does not produce any visible band (lane 3). The control PCR with primers 2 and 3 shows a band at around 500 bp (lane 4). This confirmed that it is the only species of the BKB1R mRNA. To further confirm that this is the only species of the BKB1R mRNA, Northern blots were performed on RNA isolated from IMR90 cells treated with or without 100 pg/ml IL-1. For both the IL-1 treated and untreated cells, a single band appeared at approximately 1.4 KB (Fig. 4A). This is in agreement with the calculated mRNA length (Fig. 4B). As we show in Fig. 5A, various effectors, such as TNFa, phorbol ester, bradykinin, and desArg 10kallidin, increase BKB1R mRNA. To test if these effectors induced BKB1R mRNA also contain the same 39UTR, we performed RT-PCR with the same primers as used in experiments shown in Fig. 3. As show in Fig. 5B, RT-PCR with primer 2 in all cases produced a band
FIG. 3. Reverse transcription of the BKB1R mRNA. Upper panel: Illustration of the location of the primers used in the RT reaction. Lower panel: RT reaction was carried out as described in the experimental procedure. For lane 1, RT product with primer 2 is PCR amplified with Primers 2 and 3. As control, PCR was performed with a template containing BKB1R genomic DNA and primers 2 and 3 (lane 2). For lane 3, the RT product with primer 1 is PCR amplified with primers 1 and 3. The product of control PCR with BKB1R genomic DNA and primers 1 and 3 was run on lane 4. Data represent three separate experiments.
an A at position 11 suggesting that this CA dinucleotide may be an additional recognition element for the cleavage machinery. Two closely positioned downstream U-rich elements (URE) which are often located 39 of the polyadenylation site (23) were found at 7 base and 18 base downstream of the polyadenylation site.
FIG. 4. Determine the size of the BKB1R mRNA. (A) Northern blot was performed on RNA isolated from IMR 90 cells that treated with 100 pg/ml IL-1b or vehicle for 2 h. The positions of 28 S and 18 S ribosomal RNA and the RNA size marker are indicated. (B) A schematic presentation of the BKB1R mRNA. The numbers represent the number of the bases in each section of the mRNA. The information for the 59-UTR and the coding region is obtained from our previous study (6). 32
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pGL-P and pGL-B1-39. As shown in Fig. 7B, the 39UTR of BKB1R caused the similar decrease of the luciferase activity. The luciferase activities of pGLB1-39 was 15, 24, and 8% of the wild-type pGL-P in IMRSV, IMR and Rat 1 cells, respectively. To test whether the 39-UTR of the BKB1R mRNA has a role in the regulation of the mRNA stability, we transiently transfected the pGL-P and pGL-B1-39 in the IMRSV cells. After addition of actinomycin D, RNA was isolated from the cells at several time points, and northern blot was performed as described. As showed in Fig. 8B, the mRNA for wild type luciferase pGL-P proved very stabile with a half-life of about 6h. However, the mRNA for pGL-B1-39 decayed rapidly with a half-life of approximately 1 h. DISCUSSION Most eukaryotic mRNAs have a polyadenylated (poly-A) tail at the 39 end. The addition of this tail process involves both cleavage of the primary transcript at the 39 end of the mRNA and a coupled poly-
FIG. 5. Effect of various effectors on BKB1R mRNA. (A) Northern blot was performed on RNA isolated from IMR 90 cells that were treated with 2 ng/ml TNFa, 100 nM TPA, 50 ng/ml bradykinin, 100 ng/ml desArg 10-kallidin or vehicle for 2 h. The formaldehyde gel for 18 S is shown to demonstrate equal loading. (B) RT reaction was carried out as described in the legend to Fig. 3. The primers used for each reaction are indicated. (For position of each primer, see Fig. 3, upper panel.) The RT products were amplified with primer 3 and the primer for RT reaction then run on the agarose gel.
at 500 bp but no band was produced with primer 1. This suggested that the polyadenylation site of this mRNA is not affected by these effectors. To assess the involvement of the BKB1R 39-UTR in the regulation of the mRNA stability, we created a pGL-B1-39 construct by replacing the 39-UTR of the pGL 3-P luciferase with the BKB1R 39-UTR as illustrated in Fig. 6. As controls, we also generated the pGLtailless and pGL-B1gen constructs. These constructs were transfected into the IMRSV cells. Following the transfection both Northern blot and luciferase activity assays were performed. As shown in Fig. 7A and Fig. 8A, transfection of the wild type luciferase gene (pGL3-P) produced a single band on Northern blot and high luciferase activity. The pGL-B1gen, in which a 500-bp genomic sequence of the BKB1R after the 39-UTR has been inserted into the luciferase 39-UTR, also produced a high luciferase activity. The pGLtailless, which lacks the polyadenylation signal, produced multiple bands on Northern blot and a very low luciferase activity, around 4% of the wild type. The pGLB1-39 produced a single band on Northern blot and the luciferase activity is 15% of the wild-type luciferase. This suggested that the BKB1R polyadenylation signal is functional. We also tested this 39-UTR effect on the luciferase gene in different cell line. We transiently transfected the IMRSV, IMR and Rat 1 cells with
FIG. 6. The structure of the constructs used in the study. The plasmid pGL3-P was used as the backbone to generate chimeric constructs. The essential elements of the pGL3-P are indicated. The chimeric constructs were generated as described under Experimental Procedures. The open bars represent the pGL3-P sequence elements. The closed bars represent the sequence elements of BKB1R. The numbers above the closed bars represents the location of the sequence in the BKB1R gene. The triangles represent the poly-A signal. 33
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FIG. 7. Effect of BKB1R 39-UTR on Luciferase activity. (A) IMRSV cells were transiently transfected with pGL, pGL-B1gen, pGLtailless, and pGL-B1-39. Extracts of transfected cells along with untransfected cells were prepared 48 h after transfection, and the luciferase activities were assayed with a dual luciferase system (Promega). (B) IMRSV, IMR 90 and Rat 1 cells were transiently transfected with pGL and pGL-B1-39. Extracts of transfected cells were prepared 48 h after transfection, and luciferase activities were assayed. The closed bars and the open bars represent the luciferase activity of cells transfected with pGL and pGL-B1-39 respectively. The error bars represent standard deviations of three experiments.
adenylation reaction (25). A highly conserved AAUAAA sequence located 10 to 30 nucleotides 59 of the poly-A site signals for the cleavage and poly-A reaction (26a, 26b). However, we found that an alternative AUUAAA motif which overlaps with the stop codon serves as the poly-A signal in BKB1R mRNA. This alternative motif may play an important role in the expression of this rapidly induced gene.
Posttranscriptional mechanisms are critical in regulating the expression of many rapidly induced, inflammation related genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) (13, 27, 28). We previously showed that the expression of BKB1R gene is regulated in part by mRNA stability (12). It is well accepted that the 39-UTR is the primary target for the regulation of mRNA stability (13, 14).
FIG. 8. Effect of BKB1R 39-UTR on mRNA stability of Luciferase gene. (A) The RNA were isolated from IMRSV cells that were transiently transfected with pGL, pGLtailless and pGL-B1-39 for 48 h. The Northern blot was performed with a cDNA probe for luciferase gene. The positions for 28 S and 18 S are indicated. (B) IMR90 SV40 cells were transiently transfected with pGL and pGL-B1-39. After 48 h, Actinomycin D (5 mg/ml) was added to the cultures and RNA was then isolated at 0, 1, and 3 h from pGL-B1-39 transfected cells and at 0, 3, and 6 h from pGL transfected cells. Northern blot was performed as described. The formaldehyde gel for ribosomal 28S is shown to demonstrate even loading. The Northern blots shown represent one of three separate experiments. 34
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MOLECULAR CELL BIOLOGY RESEARCH COMMUNICATIONS 8. Donaldson, L. F., Hanley, M. R., and Villablanca, A. C. (1997) Trends Pharmacol. Sci. 18, 171–181. 9. Davis, C. L., Naeem, S., Phagoo, S. B., Campbell, E. A., Urban, L., and Burgess, G. M. (1996) Br. J. Pharmacol. 118, 1469 –1476. 10. Rupniak, N. M., Boyce, S., Webb, J. K., Williams, A. R., Carlson, E. J., Hill, R. G., Borkowski, J. A., and Hess, J. F. (1997) Pain 71, 89 –97. 11. Zuzack, J. S., Burkard, M. R., Cuadrado, D. K., Greer, R. A., Selig, W. M., and Whalley, E. T. (1995) J. Pharmacol. Exp. Ther. 277, 1337–1343. 12. Zhou, X., Polgar, P., and Taylor, L. (1998) Biochem. J. 330, 361–366. 13. Ross, J. (1995) Microbiol. Rev. 59, 423– 450. 14. Ross, J. (1996) Trends Genet. 12, 171–175. 15. Agellon, L. B., and Cheema, S. K. (1997) Biochem. J. 328, 393– 399. 16. Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261–14268. 17. Shetty, S., Kumar, A., and Idell, S. (1997) Mol. Cell. Biol. 17, 1075–1083. 18. Menke, J. G., Borkowski, J. A., Bierilo, K. K., MacNei, T., Derrick, A. W., Schneck, K. A., Ransom, R. W., Strader, C. D., Linemeyer, D. L., and Hess, J. K. (1994) J. Biol. Chem. 269, 21583–21586. 19. Taylor, L., Ricupero, D., Jean, J., Jackson, B. A., Navarro, J., and Polgar, P. (1992) Biochem. Biophys. Res. Commun. 188, 786 – 793. 20. Conklin, B. R., Herzmark, P., Ishida, S., Voyno-Yasenetskaya, T. A., Sun, Y., and Bourne, H. R. (1996) Mol. Pharm. 50, 885– 890. 21. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044 –1051. 22. Sheets, M. D., Ogg, S. C., and Wickens, M. P. (1990) Nucleic Acids Res. 18, 5799 –5805. 23. Chen, F., MacDonald, C. C., and Wilusz, J. (1995) Nucleic Acids Res. 23, 2614 –2620. 24. Chou, Z.-F., Chen, F., and Wilusz, J. (1994) Nucleic Acids Res. 22, 2525–2531. 25. Wickens, M. (1990) Trends Biochem. Sci. 15, 277–281. 26a.Proudfoot, N. (1991) Cell 64, 671– 674. 26b.Molecular Biology of the Cell (1994) 3rd ed., Chap. 8, pp. 368 – 371, Garland Publishing. 27. Ristimaki, A., Garfinkel, S., Wessendorf, J., Maciag, T., and Hla, T. (1994) J. Biol. Chem. 269, 11769 –11775. 28. Hattori, Y., and Gross, S. S. (1995) Biochem. Mol. Biol. Int. 37, 439 – 445. 29. Chen, C., and Shyu, A. (1995) Trends Biochem. Sci. 20, 465– 470. 30. Myer, V. E., Fan, X. C., and Steitz, J. A. (1997) EMBO J. 16, 2130 –2139. 31. Ma, W. J., Chung, S., and Furneaux, H. M. (1997) Nucleic Acids Res. 25, 3564 –3569. 32. Ma, W., Cheng, S., Campbell, C., Wright, A., and Furneaux, H. M. (1996) J. Biol. Chem. 271, 8144 – 8151. 33. Chu, W., Presky, D. H., Swerlick, R. A., and Burns, D. K. (1994) J. Immunol. 153, 4179 – 4189. 34. Newton, R., Seybold, J., Liu, S. F., and Barnes, P. (1997) Biochem. Biophys. Res. Commun. 234, 85– 89.
Several destabilization elements, such as the AUUUA motif and stem loop structure, have been characterized in the 39-UTR (13, 29). Recently, a 36-kD RNA binding protein, HuR, which binds to the AUUUA motif with high affinity and selectivity, has been identified and cloned (30 –32). An additional mechanism for regulating the RNA stability has been proposed which involves differential polyadenylation induced by extracellular stimulation and also the production of different isoforms of 39-UTR with different effects on mRNA stability (26a, 26b, 33, 34). In this study, we show that the BKB1R 39-UTR contributes significantly to the regulation of mRNA stability. The 39-UTR which we cloned for the BKB1R mRNA proved to be only 14 bases long and does not appear to contain any known destabilization elements. However, this short 39-UTR itself may serve as a regulatory element for mRNA stability. We also show that various stimulants, such as IL-1, TNFa, phorbol ester, bradykinin and desArg 10kallidin which cause substantial increases in steady state BKB1R mRNA levels do not cause differential polyadenylation in BKB1R mRNA. Alternatively, some other regions may also be involved in the regulation of mRNA stability perhaps through direct interaction with the 39-UTR or in conjunction with some trans elements. In fact, it has been shown that the stability of urokinase-type plasminogen activator receptor mRNA is regulated in part by an element located in the coding region (17). Interestingly, two AUUUA motifs are found in the coding region of BKB1R (6, 18). It has yet to be determined if these motifs are also responsible for the regulation of BKB1 R mRNA stability. ACKNOWLEDGMENTS We thank Mr. Ned Rich at the CABR DNA/Protein Analysis Core for the expert help with sequencing and Mr. Min Zhou for the help with the 39-RACE procedure. This work was supported in part by NIH Grant HL 25776.
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