Conservation of Bmp2 Post-transcriptional Regulatory Mechanisms
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m409620200
ISSN1083-351X
AutoresDavid T. Fritz, Donglin Liu, Junwang Xu, Shan Jiang, Melissa B. Rogers,
Tópico(s)Bone Metabolism and Diseases
ResumoBone morphogenetic protein (BMP) orthologs from diverse species like flies and humans are functionally interchangeable and play key roles in fundamental processes such as dorso-ventral axis formation in metazoans. Because both transcriptional and post-transcriptional mechanisms play central roles in modulating developmental protein levels, we have analyzed the 3′-untranslated region (3′UTR) of the Bmp 2 gene. This 3′UTR is unusually long and is alternatively polyadenylated. Mouse, human, and dog mRNAs are 83–87% identical within this region. A 265-nucleotide sequence, conserved between mammals, birds, frogs, and fish, is present in Bmp2 but not Bmp4. The ability of AmphiBMP2/4, a chordate ortholog to Bmp2 and Bmp4, to align with this sequence suggests that its function may have been lost in Bmp4. Activation of reporter genes by the conserved region acts by a post-transcriptional mechanism. Mouse, human, chick, and zebrafish Bmp2 synthetic RNAs decay rapidly in extracts from cells not expressing Bmp2. In contrast, these RNAs are relatively stable in extracts from Bmp2-expressing cells. Thus, Bmp2 RNA half-lives in vitro correlate with natural Bmp2 mRNA levels. The fact that non-murine RNAs interact appropriately with the mouse decay machinery suggests that the function of these cis-regulatory regions has been conserved for 450 million years since the fish and tetrapod lineages diverged. Overall, our results suggest that the Bmp2 3′UTR contains essential regulatory elements that act post-transcriptionally. Bone morphogenetic protein (BMP) orthologs from diverse species like flies and humans are functionally interchangeable and play key roles in fundamental processes such as dorso-ventral axis formation in metazoans. Because both transcriptional and post-transcriptional mechanisms play central roles in modulating developmental protein levels, we have analyzed the 3′-untranslated region (3′UTR) of the Bmp 2 gene. This 3′UTR is unusually long and is alternatively polyadenylated. Mouse, human, and dog mRNAs are 83–87% identical within this region. A 265-nucleotide sequence, conserved between mammals, birds, frogs, and fish, is present in Bmp2 but not Bmp4. The ability of AmphiBMP2/4, a chordate ortholog to Bmp2 and Bmp4, to align with this sequence suggests that its function may have been lost in Bmp4. Activation of reporter genes by the conserved region acts by a post-transcriptional mechanism. Mouse, human, chick, and zebrafish Bmp2 synthetic RNAs decay rapidly in extracts from cells not expressing Bmp2. In contrast, these RNAs are relatively stable in extracts from Bmp2-expressing cells. Thus, Bmp2 RNA half-lives in vitro correlate with natural Bmp2 mRNA levels. The fact that non-murine RNAs interact appropriately with the mouse decay machinery suggests that the function of these cis-regulatory regions has been conserved for 450 million years since the fish and tetrapod lineages diverged. Overall, our results suggest that the Bmp2 3′UTR contains essential regulatory elements that act post-transcriptionally. Bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic protein; ARE, AU-rich element; DPP, decapentaplegic; Bt2cAMP, dibutyryl cyclic AMP; CT, Bt2cAMP and theophylline; HCNS, highly conserved noncoding sequence; nt, nucleotide; RA, retinoic acid; RACT, retinoic acid, Bt2cAMP, and theophylline; UTR, untranslated region; TNFα, tumor necrosis factor-α; RPA, RNase protection assays; CPSF, cleavage and polyadenylation factor specificity factor. were first identified by their osteogenic properties. These developmentally critical proteins are expressed widely in vertebrate embryonic structures (1Reddi A.H. J. 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For example, BMPs and their antagonists appear to play analogous, although inverted, roles in dorso-ventral axis formation in vertebrates and invertebrates (25Hogan B. Nature. 1995; 376: 210-211Crossref PubMed Scopus (37) Google Scholar). Although the BMP2 and BMP4 amino acid sequences are 91% identical and the proteins function similarly in most assays, the embryonic lethal phenotypes of null mutations proved that both genes are indispensable. The inability of BMP2 and BMP4 to compensate for each other is probably because of their distinct patterns of expression. Elucidating Bmp2 gene regulatory mechanisms is thus fundamental to understanding this crucial gene. Both Bmp2 in mouse and dpp in Drosophila are expressed in highly tissue- and stage-specific patterns. Multiple promoters and alternative splicing produce a variety of dpp transcripts in Drosophila (26St. Johnston R.D. Hoffmann F.M. Blackman R.K. Segal D. Grimaila R. Padgett R.W. Irick H.A. Gelbart W.M. Genes Dev. 1990; 4: 1114-1127Crossref PubMed Scopus (159) Google Scholar). Like Bmp2, the dpp mRNA has an unusually long 3′-untranslated region (3′UTR) with highly conserved regions (27Newfeld S.J. Padgett R.W. Findley S.D. Richter B.G. Sanicola M. de Cuevas M. Gelbart W.M. Genetics. 1997; 145: 297-309Crossref PubMed Google Scholar, 28Richter B. Long M. Lewontin R.C. Nitasaka E. Genetics. 1997; 145: 311-323Crossref PubMed Google Scholar). Our work, and that of others, suggests that mammalian Bmp2 regulation may be similarly complex (29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 30Ghosh-Choudhury N. Choudhury G.G. Harris M.A. Wozney J. Mundy G.R. Abboud S.L. Harris S.E. Biochem. Biophys. Res. Commun. 2001; 286: 101-108Crossref PubMed Scopus (60) Google Scholar, 31Heller L.C. Li Y. Abrams K.A. Rogers M.B. J. Biol. 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F9 cells lacking retinoic acid receptor-γ fail to express Bmp2 in response to RA (37Boylan J. Lufkin T. Achkar C. Taneja R. Chambon P. Gudas L. Mol. Cell. Biol. 1995; 15: 843-851Crossref PubMed Google Scholar). RA also induces the Bmp2 gene in the developing chick limb (38Francis P.H. Richardson M.K. Brickell P.M. Tickle C. Development (Camb.). 1994; 120: 209-218PubMed Google Scholar) and in medulloblastoma cells (39Hallahan A.R. Pritchard J.I. Chandraratna R.A. Ellenbogen R.G. Geyer J.R. Overland R.P. Strand A.D. Tapscott S.J. Olson J.M. Nat. Med. 2003; 9: 1033-1038Crossref PubMed Scopus (162) Google Scholar). Many Bmp2-expressing tissues (e.g. heart and cardiovasculature, limbs, central nervous system, craniofacial structures, and vertebrae) develop abnormally in vitamin A-deficient embryos or after exposure to high levels of vitamin A or other retinoids (4Hogan B. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1722) Google Scholar, 40Zile M. J. Nutr. 1998; 128: S455-S458Crossref PubMed Google Scholar). Bmp2 is expressed at three distinct levels in F9 cells. Undetectable in undifferentiated stem cells, the Bmp2 transcript is detected readily in RA-treated cells. The combination of elevated cyclic AMP levels and RA induces the Bmp2 transcript 5–6-fold more than RA alone. Cyclic AMP elevation alone induces neither differentiation nor Bmp2 expression (36Rogers M.B. Rosen V. Wozney J.M. Gudas L.J. Mol. Biol. Cell. 1992; 3: 189-196Crossref PubMed Scopus (68) Google Scholar, 41Rogers M. Cell Growth & Differ. 1996; 7: 115-122PubMed Google Scholar). The highly reproducible, differential expression of Bmp2 in F9 cells is an excellent tool for elucidating the molecular determinants controlling RA-induced Bmp2 expression. By using the F9 cell model system, we previously demonstrated that promoter function is conserved between primates and mouse (29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We now report that sequences within the Bmp2 3′UTR mediate post-transcriptional functions that have been evolutionarily conserved between mammals, birds, and fish. All plasmids were purified using Qiagen® plasmid purification kits. Dr. Peter Medveczky provided genomic DNA from chicken (Gallus gallus). Dr. Richard Pollenz provided genomic DNA from Madin-Darby canine kidney cells (dog, Canis familiaris) and CHL/IU cells (Chinese hamster, Cricetulus griseus). Dr. Jeffrey Yoder provided DNA from zebrafish (Danio rerio). Anita Antes provided genomic DNA from A549 lung cells (Homo sapiens). RNA was isolated from cell lines by using standard methods (42Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997: 4.2.3.-4.2.5.Google Scholar). Unless otherwise indicated, nucleotide positions have been provided with respect to the murine distal Bmp2 promoter that is 2,201 nt upstream of the initiator codon (ATG). All restriction and modifying enzymes were from New England Biolabs. Human—To generate an antisense probe spanning the 389 nt upstream to 146 nt downstream of the stop codon, EST clone accession number AI569017 was linearized with MscI and transcribed with T3 RNA polymerase. For the probe spanning the downstream poly(A) sites (nt 597–1,226 downstream of the Bmp2 stop codon, accession number NT_011387), a 630-bp DNA fragment was generated from genomic DNA by PCR and inserted into the EcoRI and HindIII sites of the pBluescript II KS vector (Stratagene). The sequences of the primers are as follows: HindIII-Forward, 5′CAGGAAGCTTGCAGAGTGATTGTCC3′; EcoRI-Reverse, 5′GCGAATTCAAGGTCATCATTGTAAGCG3′. This plasmid (pBShB2-3′UTR-pA2-3) was linearized with XhoI and transcribed with T7 RNA polymerase to generate antisense RNA probes. Mouse—For the mouse-specific probe spanning the stop codon and the first putative polyadenylation site, a SacI (blunted with T4 DNA polymerase) and PstI fragment was subcloned into the EcoRV and PstI site of pBluescript II KS to generate pBSB2-3′UTR-SacPst (nt 9,397–10,204). After mutating the vector AccI site, the AccI/PstI fragment spanning nt 9938–10,204 was removed; the remaining plasmid was blunted with T4 DNA polymerase and religated to generate pBSB2-3′UTR-SacI-AccI (nt 9,397–9,938). This plasmid was linearized with HindIII and transcribed with T7 RNA polymerase to generate antisense probe or with XbaI and T3 RNA polymerase to generate sense probe. For the probe spanning the downstream poly(A) sites (nt 10,202–10,781 relative to the distal promoter), a PstI/EcoRI fragment was excised from pPGLB2-3′UTR and inserted into the PstI and EcoRI sites of the pBluescript II KS vector. This plasmid (pBSB2-3′UTR-PA1-2) was linearized with XbaI and transcribed with T3 RNA polymerase to generate antisense probe and HindIII and T7 RNA polymerase to generate sense probe. RPAs—Strand-specific, [α-32P]UTP-labeled riboprobes were synthesized by using standard methods (42Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997: 4.2.3.-4.2.5.Google Scholar). RPAs were performed as in Ref. 43Strijker R. Fritz D.T. Levinson A.D. EMBO J. 1989; 8: 2669-2675Crossref PubMed Scopus (22) Google Scholar with some modifications. Briefly, radiolabeled RNA probe, total HeLa or F9 cell RNA, and yeast tRNA were co-precipitated with ethanol, denatured at 80 °C for 10 min, and hybridized overnight at 45 °C. After 20 min of RNase A/T1 digestion at 30 °C, reactions were inactivated with SDS and proteinase K, followed by phenol/chloroform extraction. Subsequently, RNAs were ethanol-precipitated, dissolved in 15 μl of gel loading buffer, and electrophoresed on denaturing 5% polyacrylamide gels (8 m urea; 37.5:1, acrylamide:bisacrylamide). Protected RNAs were visualized by using autoradiography and quantified using an Amersham Biosciences PhosphorImager and ImageQuant software. Genomic clones were obtained by PCR using a forward primer to a region just upstream of the stop codon that is identical or nearly identical in all vertebrates (5′CAGGACATGGTGGTGGAGGG3′) and species-specific reverse primers. The mammalian and chick products used a primer identical to the chick sequence (5′GCACTTTGCCATAGTAACCTTCC3′). The zebrafish primer was 5′GCCTTCAGCATGTTATATCATGAC3′). PCRs contained 1 unit of Taq recombinant DNA polymerase (Roche Applied Science), 100 ng of genomic DNA, 50 pmol of each primer, 0.25 mm dNTPs, 1.5 mm MgCl2, and buffer conditions as recommended by the manufacturer (Roche Applied Science). 100-μl reactions were incubated as follows in a Mastercycler gradient PCR machine (Eppendorf): denature 97 °C/5 min, anneal 47 °C/30 s (53 °C zebrafish), extend 72 °C/3 min followed by 35 cycles using a 94 °C denaturation. The PCR products were separated by PAGE (37.5:1 acrylamide:bisacrylamide), visualized by ethidium bromide staining, and confirmed by Southern blotting (42Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997: 4.2.3.-4.2.5.Google Scholar) using the murine highly conserved noncoding sequence (HCNS, Fig. 1A) as probe. PCR fragments (∼500 bp) were purified by gel electrophoresis, passively eluted from a gel slice, and cloned into the pCRII TA cloning vector (Invitrogen) according to the manufacturer's instructions. Clones from all species were subcloned into the EcoRI site pGEM-4 (Promega) for in vitro transcript synthesis. The University of Medicine and Dentistry of New Jersey Medical School Molecular Resource Facility (Newark, NJ) sequenced each plasmid by using T7 and Sp6 primers. The accession numbers and region cloned were human AL035668, 131,066–131,576; mouse AL831753, 133,140–133,650, chicken accession numbers BU423990, 63–540; and zebrafish accession numbers AL929237, 88,314–88,749. Newly cloned hamster (AY722409), dog (AY722408), and cow (AY714781) sequences have been submitted to GenBank™. Our cloned dog sequence matches the recently completed canine genomic sequence AAEX01031455. Construct A (nt –1,237 to 471, pGL1.7XX)—See Ref. 29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar for construct A. pBSB2-3′UTR (nt 9,392–11,604)—A 6-kb BamHI/NotI fragment was subcloned from phage B2 (31Heller L.C. Li Y. Abrams K.A. Rogers M.B. J. Biol. Chem. 1999; 274: 1394-1400Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) creating mB2-BamI (nt 5,102–11,604). pBSB2-3′UTR was created by excising a SacI fragment containing nt 5,102–9,392 and religating. pGL2BasicΔBam+Sac—QuikChange™ site-directed mutagenesis (Stratagene) was used to add a SacI/SstI site at nt 2100 of plasmid pGL2BasicΔBam (29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The sequences of the mutating oligomers used are as follows: Sac-Forward, 5′-GAGGAAAACCTGTTGAGCTCAGAAGAAATGCCATCTAGTG-3′; Sac-Reverse, 5′-CACTAGATGGCATTTCTTCTGAGCTCAACAGGTTTTCCTC-3′. Construct B (nt –1,237 to 471 and nt 9,392–11,604, pGLB2–5′3′)— Plasmid pBSB2-3′UTR (nt 9,392–11,604) was digested with SmaI and SacI. A 2,212-bp fragment containing the Bmp2 stop codon, 3′UTR, and downstream sequence was cloned into pGL2BasicΔBam+Sac digested with StyI (blunted with Klenow in the presence of dNTPs) and SacI to create pGLB2–3′UTR. A DraIII and XbaI fragment containing the 1,702-bp Bmp2 promoter was excised from construct A (29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and was inserted in place of the 2,381-nt DraIII and XbaI fragment in pGLB2–3′UTR. Construct C (nt –1,237 to 471 and nt 9,574–10,204, pGLB2–5′3′CNS)—Plasmid pGLB2–3′UTR was digested by PstI and BamHI, blunted with T4 DNA polymerase in the presence of dNTPs, and religated to remove the fragment between nt 10,204 and 11,444 creating pGLB2–3′UTRΔPstBam. Plasmid pGLB2–3′UTRΔPstBam was cut with PvuII and SacI, blunted by T4 DNA polymerase in the presence of dNTPs, and the ends religated to create pGLB2–3′UTRCNS. This deletion leaves all of the highly conserved sequences. The Bmp2 promoter fragment from construct A was inserted upstream of luciferase as described for construct B. Construct D (nt –1,237 to 471 and nt 9,392–11,604, pGLB2–5′SVpA-3′UTR)—The 2,212-bp Bmp2 fragment from pBSB2-3′UTR was excised with SacI (blunted with T4 DNA polymerase in the presence of dNTPs) and SalI and then cloned into pGL2Basic cut with BamHI (filled in with T4 DNA polymerase) and SalI to create pGLB2-SVpA-3′UTR. The Bmp2 promoter fragment from construct A was inserted upstream of luciferase as described for construct B. Construct E (nt –1,237 to 471 and nt 10,202–11,604, pGLB2–5′3′ΔSacPst)—To remove the 810-bp fragment between nt 9,392 and 10,202, pGLB2–3′UTR was cut with SacI and PstI, blunted with T4 DNA polymerase in the presence of dNTPs, and religated to create pGLB2–3′UTRΔSacPst. The Bmp2 promoter fragment from construct A was inserted upstream of luciferase as described for construct B. pGemB2-KA (nt 9,455–9,938) and plasmids containing the homologous region from human, chick, and zebrafish were synthesized by PCR as described above and linearized with BamHI. SacI/PstI (nt 9,397–10,202) or PvuII/PstI (9,574–10,202) fragments obtained from pBS-3′UTR were cloned into SacI/PstI or SmaI/PstI-digested pGem4 to make pGBmp2-SacPst and pGBmp2-PvuIIPst, respectively. These plasmids were linearized with AccI to make sense probes spanning nt 9,397–9,938 or 9,574–9,938, respectively. pGBmp2-SacPst was digested with PvuII and RsaI to make pGBmp2-PvuIIRsa (nt 9,574–9,735) or RsaI and AccI (blunted with T4 DNA polymerase) to make pGBmp2-RsaAcc (nt 9,735–9,938). These plasmids and wild type and mutated TNFα plasmids (44Mukherjee D. Gao M. O'Connor J.P. Raijmakers R. Pruijn G. Lutz C.S. Wilusz J. EMBO J. 2002; 21: 165-174Crossref PubMed Scopus (310) Google Scholar) were linearized with HindIII. All linearized plasmid templates were transcribed with SP6 RNA polymerase F9 embryonal carcinoma cells were plated on dishes pre-coated with 1% gelatin and incubated at 37 °C with 10% CO2. The culture media consisted of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum and 2 mm glutamine. The cells were induced to differentiate into parietal endoderm by adding 1 μm all-trans-retinoic acid, 250 μm Bt2cAMP, and 500 μm theophylline (RACT). Undifferentiated control cells were treated with 250 μm Bt2cAMP and 500 μm theophylline (CT). Transfections were performed essentially as described by Vasios et al. (45Vasios G.W. Gold J.D. Petkovich M. Chambon P. Gudas L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9099-9103Crossref PubMed Scopus (250) Google Scholar). Briefly, for 96-h drug treatments, F9 cells were plated at 1 × 106 or 0.3 × 106 (CT only) cells per 100-cm dish (Nunc) for 12 h, drugged for 48 h with CT or RACT, transfected by overnight calcium phosphate precipitation, and then cultured for an additional 24–48 h with drugs. Each 100-cm dish was co-transfected with 10 μg of reporter plasmid and 3 μg of pβAclacZ (45Vasios G.W. Gold J.D. Petkovich M. Chambon P. Gudas L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9099-9103Crossref PubMed Scopus (250) Google Scholar) containing the β-galactosidase coding region driven by the constitutive β-actin promoter. Cells were extracted, and luciferase activity was determined by using the Promega Luciferase Assay System and a Monolight 2010 luminometer (Analytic Luminescence Laboratory). Luciferase activity was normalized for transfection efficiency by dividing the raw luciferase value by the units of β-galactosidase activity (1 unit = A1 420·μl–1·h–). Sequences were subcloned into the pGEM4 (Promega) polylinker downstream of the SP6 promoter and upstream of the HindIII site. After HindIII digestion, plasmids were transcribed with SP6 RNA polymerase with 7MeGpppG and [α-32P]UTP. The design of these transcripts mimics that of transcripts used extensively for this purpose (44Mukherjee D. Gao M. O'Connor J.P. Raijmakers R. Pruijn G. Lutz C.S. Wilusz J. EMBO J. 2002; 21: 165-174Crossref PubMed Scopus (310) Google Scholar, 46Ford L.P. Wilusz J. 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The Bmp2 3′UTR Is Unusually Conserved—Surveys of conserved regions in 3′UTRs defined "highly conserved" as sequences of >100 nt with 70% identity conserved for 300 million years of evolution (50Duret L. Dorkeld F. Gautier C. Nucleic Acids Res. 1993; 21: 2315-2322Crossref PubMed Scopus (140) Google Scholar, 51Shabalina S.A. Ogurtsov A.Y. Lipman D.J. Kondrashov A.S. Nucleic Acids Res. 2003; 31: 5433-5439Crossref PubMed Scopus (18) Google Scholar, 52Spicher A. Guicherit O.M. Duret L. Aslanian A. Sanjines E.M. Denko N.C. Giaccia A.J. Blau H.M. Mol. Cell. Biol. 1998; 18: 7371-7382Crossref PubMed Scopus (56) Google Scholar). Because 265 nt of the Bmp2 3′UTR is 73% conserved between mammals and fish (450 million years of separation), this region fits within this class (Fig. 1) (29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). For brevity, we have termed the Bmp2 highly conserved noncoding sequence the "HCNS." A more comprehensive analysis of Bmp2 3′UTRs from more evolutionarily separated mammals indicates that the entire 3′UTR including the polyadenylation (poly(A)) signals is unusually conserved. To analyze the rest of the 3′UTR, we aligned Bmp2 sequences from our PCR-generated clones and newly deposited GenBank™ sequences from additional mammals using the MultiPipMaker global sequence alignment program (see site bio.cse.psu.edu/pipmaker/) (Fig. 1). The 3′UTRs of the human and dog Bmp2 transcripts are 86% identical over 1,217 nt and 83% identical over 1,088 nt, respectively, to the mouse transcript. Between Bmp2 genes from four mammalian orders (Rodentia, Primates, Carnivores, and Artiodactyls), the highly conserved block is 95% identical over 370 nt. Furthermore, the striking conservation of the entire 3′UTR between several rodents, humans, and chimpanzees and dog, deer, and cow implies vital post-transcriptional regulatory functions for the whole 3′UTR. The Bmp2 and Bmp4 3′UTRs Differ—The BMP2 and BMP4 proteins are 91% identical and have similar biological activities but are expressed in different patterns (4Hogan B. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1722) Google Scholar, 53Lyons K.M. Pelton R.W. Hogan B.L.M. Devlelopment (Camb.). 1990; 109: 833-844PubMed Google Scholar, 54Lyons K. Pelton R. Hogan B. Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (401) Google Scholar, 55Jones C.M. Lyons K.M. Hogan B.L.M. Development (Camb.). 1991; 111: 531-542PubMed Google Scholar). Our analyses of ESTs and mRNA sequences indicate that human and mouse Bmp4 mRNAs end at a single poly(A) signal resulting in 293 or 336 nt 3′UTRs, respectively. We attempted to align Bmp2 and Bmp4 3′UTR sequences from many species; however, the Bmp2 HCNS is clearly absent in all Bmp4 genes (see Ref. 29Abrams K.L. Xu J. Nativelle-Serpentini C. Dabirshahsahebi S. Rogers M.B. J. Biol. Chem. 2004; 279: 15916-15928Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar and data not shown). We also attempted to align vertebrate Bmp4 3′UTRs to themselves, but Bmp4 3′UTRs are poorly conserved relative to the coding region. BMP2 and BMP4 form a subgroup with the invertebrate DPP protein. Protostomes and other deuterostomes such as cephalochordates, hemichordates, urochordates, and echinoderms appear to have only one Bmp2/4/dpp-like gene (22Lelong C. Mathieu M. Favrel P. Biochimie (Paris). 2001; 83 (and references therein): 423-426Crossref PubMed Scopus (23) Google Scholar, 56Panopoulou G.D. Clark M.D. Holland L.Z. Lehrach H. Holland N.D. Dev. Dyn. 1998; 213: 130-139Crossref PubMed Scopus (72) Google Scholar). Dpp 3′UTRs from four Drosophila species that diverged 40–80 million years ago are perfectly conserved for 110 nt (27Newfeld S.J. Padgett R.W. Findley S.D. Richter B.G. Sanicola M. de Cuevas M. Gelbart W.M. Genetics. 1997; 145: 297-309Crossref PubMed Google Scholar, 28Richter B. Long M. Lewontin R.C. Nitasaka E. Genetics. 1997; 145: 311-323Crossref PubMed Go
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