250
Biochimica et Biophvsica Acta, ll187 (19901 25(/-252 Elsevier
BBAEXP 90190
B B A Report - Short Sequence-Paper
Manganese superoxide dismutase: nucleotide and deduced amino acid sequence of a c D N A encoding a new human transcript Susan L. Church Department of Pediatrics, St. Louis Children's Hospital, Washington Unil;ersity School of Medicine, St. Louis, M I t U.S.A.)
(Received 29 June 1990)
Key words: Superoxide dismutase; Manganese enzyme: eDNA cloning; Amino acid sequence; Gene expression; (Human)
Three human cDNA libraries were screened with a human manganese superoxide dismutase (Mn-SOD) cDNA under moderately stringent conditions to characterize a large 4 - 6 kb RNA species which hybridizes to Mn-SOD in RNA blot analyses. A new 4.2 kb Mn-SOD cDNA clone (Mn-SOD 1) was isolated. Its long 3426 nucleotide 3'-untranslated sequence contains both of the 240 base 3'-untranslated sequences of the 1 kb Mn-SOD 4 and 5 cDNAs. This is a fully processed, cytoplasmic RNA species and raises the possibility of a role for particular 3'-untranslated sequence selection in Mn-SOD gene regulation.
The manganese-containing superoxide dismutase (Mn-SOD) is one member of a family of metalloenzymes which are important in cellular antioxidant defense. In eukaryotes, Mn-SOD is encoded by a nuclear gene and is found predominantly in the mitochondria. Messenger R N A heterogeneity of Mn-SOD exists, as evidenced by the presence of different human cDNAs of 1 kb [1-4] and by the consistent and unexplained presence of longer hybridizing transcript at 4 [2] or 6 kb [1]. In the present study, to further clarify the molecular heterogeneity of the human Mn-SOD transcripts, three human c D N A libraries were screened with a Mn-SOD coding region cDNA. Three full-length and two truncated clones were isolated and fully sequenced. An 827 bp insert from phMnSOD4 (vector-pBR327) obtained from Bell [5] was 32p-labeled using random primer labeling [6] and then used to screen two human placental libraries constructed in vector ?,gtll (obtained from E. Sadler and J.D. Gitlin) and a human liver library constructed in the vector Lambda Zap (obtained from R.T. Wetsel). Three cDNAs from the first placental library (4.2, 1.8 and 1.5 kb), two from the second placental library (3.8 and 1.0 kb) and a 1 kb
Abbreviations: SOD, superoxide dismutase; Mn, manganese; kb, kilobase; EDTA, ethylenediaminetetraacetic acid. The sequence data in this paper have been submitted to the EMBL/Genbank under the accession number M34665. Correspondence: Department of Pediatrics, Box 8116, Washington University School of Medicine, 400 S. Kingshighway Blvd., St. Louis, MO 63110, U.S.A.
insert from the liver library were fully sequenced using the double-stranded dideoxy chain termination method [7]. Polylinker and unique internal restriction endonuclease sites and the Erase-a-Base (Sigma) nested deletion system were employed to subclone and sequence the larger clones. Sequence confirmation was achieved by using oligonucleotide primers to read the opposite strand, or by verification of multiple overlapping sequences produced by nested deletions. To confirm that the 4.2 kb transcript was fully processed and located in the cytoplasm, total cellular R N A isolated by acid guanidinium thiocyanate-phenol-chloroform extraction method [8] and total cytoplasmic R N A [9] were enriched for poly(A) + R N A by affinity chromatography on oligo(dT)-celhilose [10]. 10 /~g of total cellular or cytoplasmic RNA, or 2 ~g of poly(A) + RNA was size fractionated on a 1% agarose, 200 mM 4-morpholinepropanesulfonic acid (pH 7.4), 1 mM EDTA and 18% formaldehyde gel, transferred to nylon (Hybond, Amersham) and hybridized to 32p-labeled antisense R N A transcribed from Sp6 or T7 (Promega). The blot was washed and exposed to X A R film at - 7 0 o C. The entire nucleotide sequences for the unique MnSOD cDNAs are depicted in Fig. 1. Dramatic sequence differences are found in the 3'-untranslated sequences and the polyadenylation signals. The longest Mn-SOD c D N A (Mn-SOD 1) contains within its sequence, both of the 240 nucleotide 3' untranslated cassettes of the short Mn-SOD cDNAs 4 and 5. It neglects the AGTAAA polyadenylation signal [11] of Mn-SOD 4 (which is almost identical to the shorter Mn-SOD cDNAs described by Wispe [2] and Ho [1]) and utilizes
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
251 GATCTGGTCGACCTGGCTACCGGCTTCGGCAGCGGCTTCAG~AGATCGGCGGCATCAGCGGTAGCACCAGCACTAGCAGCATGTTGAGCCGGGCAGTG 98
MetLeuSerArgAlaWal TGCGG•A•CAGCAGGCAGCTGGCTCCGG•TTTGGGGTATCTGGGCTC•AGGCAGAAGCA•AG•CT•C•CGACCTGCCCTACGACTACGGCGCCCTGGAAC•T CysG~YThrSerArgG~nLeuA~aPr~A~aLeuG~yTyrLeuG1ySerArgG~nLYsHisSerLeuPr~AspLeu~r~TyrAspTyrG~yA~aLeuG1uPr~
200
CACATCAACGCGCAGATCATGCAGCTGCACCACAGCAAGCACCA~GCGGCCTACGTGAACAACCTGAACGTCACCGAGGAGAAGTACCAGGAGGCGTTGGCC HisIleAsnA•aG•nI•eMetG•nLeuHisHisSerLysHisHisA•aA•aTyrVa•AsnAsnLeuAsnVa•ThrG•uG•uLysTyrG•nG•uA•aLeuA•a
302
AAGGGAGATGTTACAGCCCAGATAG~TCTTCAG~CTG~ACTGAAGTT~AATGGTGGTGGTCATATCAATCATAGCATTTTCTGGACAAACCTCAGCCCTAAC LysG•yAspVa•ThrA•aG•nI•eA•aLeuG•nPr•A•aLeuLysPh•AsnG•yG•yG•yHisI••AsnHisS•rI•ePheTrpThrA•nLeuSerPr•Asn
404
GGTGGTGGAGAAC••AAAGGGGAGTTG•TGGAAGC•ATCAAACGTGA•TTTGGTT•CTTTGA•AAGTTTAAGGAGAAG•TGA•GG•TGCATCTGTTGGTGT• 506 G~yG~yG~YG~uPr~Ly~G~yG~uLeuLeuG~uA~aI~eLysArgAspPheG~yS~rPheAs~LysPheLysG~uLysLeuThrA~aA~aSerVa~G~yVa~ CAAGGCTCAGGTTGGGGTTGGCTTGGTTTCAATAAGGAACGGGGA•A•TTACAAATTGCTG•TTGT••AAAT•AGGAT••A•TG•AAGGAA•AA•AGGC•TT 608 G•nG•ySerG•yTrpG•yTrPLeuG•yPheAsnLysG•uArgG•yHisLeuG•nI•eA•aA•aCysPr•AsnG•nAspPr•L•uG•nG•yThrThrG•yLeu ATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTACTACCTTCAGTATAAAAATGTCAGGCCTGATTATCTAAAAGCTATTTGGAATGTAATCAACTGG I~ePr~LeuLeuG~yI~eAsPva~TrpG~uHi~A~aTyrTyrLeuG~nTyrLYsA~nVa~ArgPr~AspTyrLeuL~A~aI~eTr~AsnVa~I~eAsnTrp GAGAATGTAACTGAAAGATACATGGCTTGCAAAAAGTAA GluAsnValThrGluArgTyrMetAlaCysLysLys M~ SOD Mn SOD Mn SOD
1 ACCACGATCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAG 4 ACCACGATCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAG 5 A C C A C G A T C G T T A T G C T G A .......
1 TGGTATAGAGTACTGCAGAATACAGTAAGCTGCTCTATTGTAGCATTTCTTGATGTTGCTTAGTCACTTATTTCATAAACCAACTTAATGTTCTGAATCA
710
800
900
4 TGGTATAGAGTACTG•AGAATA•AGTAAG•TGCTCTATTGTAG•ATTTCTTGATGTTGCTTAGTCACTTATTTCATAAA•CAACTTAATGTT•TGAATCA I ATTTCTTA•TAAACATTTTGTTATTGGGCAAGTGATTGAAAATAGTAAATGCTTTGGTGGTGGATTGAAACTCTGAATTGGGACATTTTCTTCAGAGAGC 4 ATTTCTTACTAAACATTTTGTTATTGGGCAAGTGATTGAAAAAAAAAAAAAAAAAAAAAA TAAATTACAACTTTGTCATTTTATAAAACCATCAAAAATATT•CATCCATATATACTTTGGGGACTTGTAGGGATGCCTTTCTAGTCCTATTCTATTGCA
i000 ii00
GTTATAGAAAATCTAGT•TTTTG••••AGTTACTTAAAAATAAAATATTAACACTTTCCCAAGGGAAA•ACTCGGCTTTCTATAGAAAATTG•ACTTTTT 1200 GTCGAGTAATTCTGCAGTGATACTTCTGGTAGATGTCACCCAGTGGTTTTTGTTAGGTCAATGTTCCTGTATATAGTTTTTG•AAATAGAG•TGTATA•T 1300 GTTTAAATGTAGCAGGTGAACTGAACTGGGGTTTGCTCACCTGCACAGTAAAGGCAAACTTCAACAGCAAAACTGCAAAAAGGTGGTTTTTGCAGTAGGA GAAAGGAGGATGTTTATTTGCAGGGCG••AAGCAAGGAGAATTGGG•AGCTCATGCTTGAGACC•AATCTCCAGATGATGACCTACAAGCTAGGATATTT AAAGGCAGTGGTAAATTTCAGGAAAGCAGAAGTTAAAGGCAAAATTGTAAATCAGTCGAGACTGGGTGCCTTCAGGGTGGTATGGCTAGTATCA••AAAA TTGTAAATCACTACATGAAGCTTATATATTGGTTTGGCCTGA~AGGTGAAGTGGGTAGGCAGGGGGCGGGCTTACAGGTTATGGTGGATT~AAAGACTCC
CTGATTTGTGATTGGTTAAGGAAGCAAAGCTTTGTCTAAAAA•TTGGGTc•G•GAGAAGGAA•AATAGT•TG••AGC•CCTCAGGAAAGAA•A•TGAGAGC AAAGAATGGAGGTCAGAGTTAGTCCCTGGTGTTCCCCCTTATCTGACGTCTGTGTGAATCCCATTTGGTGGGGGTCTGGGTTTCTGAAAAGCTAGCTCAG GGGCA~GTGTTAAGGATGT~TCTAGGTGACTCTAACTT~CCTGGCTATTGTTTGAAACTGTTATGACCTTCTTGCTTATCAGCTTGCTGGTTTCCTTCTC GGGGCGAGCTGGGTGCCTGGAGTTTTCGGTGAAGGAAACTCAAGATTCTCCTTTATTTCTGTGCTTGTGGGAAT~cTGcA~cAAAGAGGGGTCCC TGACTAC~GT~TCACAGGGATACTTTTTGTATATTTGGCTTAGCATTCATACATTTTGGCCATGTTGGTTTCCATCCATCTGGCCTAATTTACTGTTTTT
GAATATTTCCATTTGTTT•TAAATGTAA•TAcAGATAATAG•ATGGGGTGAGCAA•ATCTGATGTA•ATAGGTTTATCTCCTATTGGAATATTTTCTTTA TATAGGCGTTTTTTTTTTTTCTTTTTTTTTGGAGACAGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCG•GACcGGAG•TcAcTG•AA••T•cA
~TT~C~GGGTT~AAGTGATTGT~CCACCTCAGCCTCCTGAATAG~TGGGATTATAGGTGCATGCTACCATGCCTGGCTACTT~TTGTATTTTTAGCAGAG ACAGGGTTTCACCATGTTGG•CAGGGTGGTCTCGAA•TCCTGAC•T•AAGTGAT••GT•TGGCTCAGCCTCCCAAATGCTGGGATTACAGGTGTGAGCCA CTGCACCTGGCCTATATAGGCTTTTTTCTTAAACCTATTTAGTAATGTTTTCCCAAGTTTATTTTTTATTTTTAATTTTTTTCCCCAAGTTTATTTTTCT ATTTTTTTTTCATGGAAAATGGGGTAACTTAGCAGTTTCAATATTGAAGACTGAAGTTTAAAAAAAAATTTAAATTCAAGGTACTTTTAAATTTCAGTTA GGAAAAGTAGGGCTTTAAA-~TTATTAGAGACAAGTACCAAAGCGGTGTGTGTATGTGTGTGTGTGTATGCATGCTTGTGGATTGGA~AAACTTTGGAGAC
TGATTACTTTTCATTATATATGTGTCACAGTGAAACAGCTTTTATGTGTCATGTAAGATTACTGCTTGCCTCTCTAAGGAAGGTCGTGACTGTTTAAATA GACGGGCAAGGTGGAA~TTTTGAAAGATGAGCTTTTGAATATAAGTTGTCTGCTAGATCATGGTTTGTATTGAACTAACAAGGTTTGCAGATCTGCTGA CTTATATAAGCTTTTTGATTCCTACTAAGCTTTAAGATTTAAAAAATGTTCAATGTTGAAATTTCTGTGGGGCTCTATTTTTG•TTTGGcTTT•TGGGTG
AGAGAGTGAGGAAGCATTCTTTCCTTCACTAAGTTTGT~TTTCTTGTCTTCTGGATAGATTGATTTTAAGAGACTAAGGGAATTTACAACTAGCGCTTTT AGTCCATCTGGTGGGAAAGGAGACTGTAGATTGTTAGGGCTGGG•GGGTGA•TCACAT•TGTAAT•CcCAGCACTTTGGGAGGCCAAGGCAGG•AGAACA CTTGAAGGAGTTCAAGACCAGCCGTGGCCCAACGTGGTGAAACCCTGTCTCTACTAAAAATACCAAGAAATTGTTTAGCTCTGTTTTTCATATAGAAAAT AGAAAAGGTAAATTCCGGTTTTT•TTCTGAAAAGAACAAGTATTGTTCATCCAAGAAGGGTTTTTGTGACTGAATCAGCAGTGCCTGCCCTAGTATAGTG
TGCTTCAAAAACCTCAGCATGATTAGTGTTGGAGCAAAACAAGGAAGCAAAGCAAATA•AGTTTTTGAAATTCTAT•TGTTG•TTGAA•TATTTTGTAAT AATTAAACTTTGATGTTGAGAAATCACAACTTTATTGTACACTTCATTGCAACTTGAAATTCATGGTCTTAAAAGTGAGATTGAATTTCTATTGAG~G~C
TTTAAAAAGTAATACCCAAA•CATAAAGGTTAAAATCTATGTATATTGAGTcATATCTAAACCACGGTATAAACATAAATTGTATTTCCTGTTTTAATTC 1 AGGGGAAGTACTGTTTGGGAAAGCTATTATTAGGTAAATGTTTTACAAATTACTGTTTCTCA•TTT•AGTCATAC••TAATGATCCCAGCAAGATAATGT Mn SOD
5
1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000
............ T C A T A C C C T A A T G A T C C C A G C A A G A T A A T G T
1 CCGTCTCTTCTAAGATGTCATCAAGCCTGGTACATACTAAAACCCTATAAGGTCCTGGATAATTTTTGTTTGATTATT•ATTGAAGAAA•ATTTATTTTC
4100
5 •CGT•T•TT•TAAGATGTCATCAAG•CTGGTACATACTAAAACCCTATAAGGTCCTGGATTAATTTTGTTTGATTATTCATTGAAGAAACATTTATTTTC 1 CAATTGTGTGGAAGTTTTTGACTGTTAATAAAAGAATCTGTCCAACCCAATCGGTAGGAGTCGACAGGATGAATTC 5 CAATTGTGTGGAAGTTTTTGACTGTTAATAAAAGAATCTGTCCAACCC
4176
Fig. 1. The DNA and derived proton sequence of human Mn-SOD cDNAs. The Y-untranslated re~ons of Mn-SOD 1, 4 and 5 ~e depicted. Coding re,on nucleoOde substitutions are denoted by astefieks and described in the text. The human Alu repetitive sequence element is indicated by underlimng. the A A T A A A polyadenylation signal of M n - S O D 5 (which completes a different shorter M n - S O D c D N A sequence reported by Beck [3]). M n - S O D 1 contains a consensus splice junction sequence with the invariant G T splice d o n o r and A G splice accepter sequences [12,13] at requisite positions to be able to produce
M n - S O D 5 f r o m M n - S O D 1. Bases 2659-2702 comprise an AT-rich sequence [ T T A T T T r [ T T A T ] T T T T A A T ] T T T . . . . [ T T A T T T T C T A T ] , similar to AU-rich sequences previously described in the Y-untranslated regions of several lymphokines, cytokines and protooncogenes m R N A s which are transiently expressed [14]
252 I
28S
2
3
4
-
region of this new Mn-SOD 1 cDNA and almost identical coding regions, including the mitochondrial leader sequences, raises the possibility of a regulatory role for Mn-SOD enzyme biosynthesis by the selection of particular Mn-SOD 3'-untranslated region cassettes.
18S -
Acknowledgements
Fig. 2. RNA blot analysis of total and cytoplasmic term placental RNA. Total (1) and cytoplasmic (2) RNA was prepared from term human placental RNA and poly(A)-enriched from total (3) and cytoplasmic (4) term placental R N A and separated by agarose gel electrophoresis. RNA was transferred to Nylon and hybridized to 32P-labeled antisense Mn-SOD 5 RNA. In both total and cytoplasmic human term placental RNA, there are two hybridizing bands, one at 4.2 kb and one at 1 kb. Poly(A) selection enriches the smaller Mn-SOD transcript. The large 4.2 kb Mn-SOD transcript is,found as a processed message abundantly in the cytoplasm.
and which have been implicated as mRNA-destabilizing sequences [15]. Mn-SOD 1 also contains, in its complementary strand, a sequence 84% homologous to an Alu repetitive element [16] (depicted by underlining) which is flanked by a 9 base repeat. As can be seen in Fig. 2, this large transcript is not heterologous nuclear unprocessed R N A (which can contain Alu repeats [16]) but exists abundantly in the cytoplasm as a processed R N A species. Additional sequence differences in these Mn-SOD cDNAs from the Mn-SOD sequences previously reported [1-4] include a C to T substitution at nucleotide 325 which results in a Thr to Ile amino acid substitution in all clones. Mn-SOD 4 contains an A to C substitution at base 159 which results in a Ser to Arg amino acid substitution and Mn-SOD 5 has a C to T substitution at base 198 causing a Pro to Leu substitution. Recognition of the co-existence of multiple human Mn-SOD transcripts, with dramatic sequence differences in the additional 3426 bases of 3'-untranslated
Many fruitful discussions and advice were provided by Drs. Harvey Colten, Arnold Strauss and James Grant. S.L. is a recipient of Clinical Investigator Award K8HD000885A from the National Institutes of Health. References I Ho, Y. and Crapo, J.D. (1988) FEBS 229, 256-260. 2 Wispe, J.R., Clark, J.C., Burhars, M.S., Kropp, K.E., Korfhagen, T.R. and Whitsett, J.A. (1989) Biochim. Biophys. Acta 994, 30-36. 3 Beck, Y., Oren, R., Amit, B., Levanon, A., Gorecki, M. and Hartman, J.R. (1987) Nucl. Acids Res. 15, 9076. 4 Heckl, K. (1988) Nucl. Acids Res. 16, 6224. 5 Xiang, K., Cox, N.J., Hallewell, R.A. and Bell, G.I. (1987) Nucl. Acids Res. 15, 7654. 6 Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 7 Sanger, F., Nickleu, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 8 Beyarsky, A., Vinogradova, T. and Rajewsky, K. (1989) Nucl. Acids Res. 17, 2920-2932. 9 Benoff, S. and Nadal-Ginard, B. (1979) Proc. Natl. Acad. Sci. USA 76, 1853-1857. 10 Davis, L.G., Dibner, M.D. and Battey, J.F. (1986) in Methods in Molecular Biology (Davis, L.G., Dibner, M.D. and Battey. J.F., eds), pp. 139-142, Elsevier, New York. 11 Birnsteil, M,L., Busslinger, M. and Strub, K. (1985) Cell 41, 349-359. 12 Cech, T.R. (1983) Cell 34, 713-716. 13 Sharp, P.A. (1981) Cell 23, 643-646, 14 Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S. and Cerami, A. (1985) Proc. Natl. Acad, Sci. USA 83, 1670-1674. 15 Shaw, G. and Kamen, R. (1986) Cell 46, 659-667. 16 Jelinek, W.R. (1982) Annu. Rev. Biochem. 51, 813-844.
Biochimica et Biophvsica Acta, ll187 (19901 25(/-252 Elsevier
BBAEXP 90190
B B A Report - Short Sequence-Paper
Manganese superoxide dismutase: nucleotide and deduced amino acid sequence of a c D N A encoding a new human transcript Susan L. Church Department of Pediatrics, St. Louis Children's Hospital, Washington Unil;ersity School of Medicine, St. Louis, M I t U.S.A.)
(Received 29 June 1990)
Key words: Superoxide dismutase; Manganese enzyme: eDNA cloning; Amino acid sequence; Gene expression; (Human)
Three human cDNA libraries were screened with a human manganese superoxide dismutase (Mn-SOD) cDNA under moderately stringent conditions to characterize a large 4 - 6 kb RNA species which hybridizes to Mn-SOD in RNA blot analyses. A new 4.2 kb Mn-SOD cDNA clone (Mn-SOD 1) was isolated. Its long 3426 nucleotide 3'-untranslated sequence contains both of the 240 base 3'-untranslated sequences of the 1 kb Mn-SOD 4 and 5 cDNAs. This is a fully processed, cytoplasmic RNA species and raises the possibility of a role for particular 3'-untranslated sequence selection in Mn-SOD gene regulation.
The manganese-containing superoxide dismutase (Mn-SOD) is one member of a family of metalloenzymes which are important in cellular antioxidant defense. In eukaryotes, Mn-SOD is encoded by a nuclear gene and is found predominantly in the mitochondria. Messenger R N A heterogeneity of Mn-SOD exists, as evidenced by the presence of different human cDNAs of 1 kb [1-4] and by the consistent and unexplained presence of longer hybridizing transcript at 4 [2] or 6 kb [1]. In the present study, to further clarify the molecular heterogeneity of the human Mn-SOD transcripts, three human c D N A libraries were screened with a Mn-SOD coding region cDNA. Three full-length and two truncated clones were isolated and fully sequenced. An 827 bp insert from phMnSOD4 (vector-pBR327) obtained from Bell [5] was 32p-labeled using random primer labeling [6] and then used to screen two human placental libraries constructed in vector ?,gtll (obtained from E. Sadler and J.D. Gitlin) and a human liver library constructed in the vector Lambda Zap (obtained from R.T. Wetsel). Three cDNAs from the first placental library (4.2, 1.8 and 1.5 kb), two from the second placental library (3.8 and 1.0 kb) and a 1 kb
Abbreviations: SOD, superoxide dismutase; Mn, manganese; kb, kilobase; EDTA, ethylenediaminetetraacetic acid. The sequence data in this paper have been submitted to the EMBL/Genbank under the accession number M34665. Correspondence: Department of Pediatrics, Box 8116, Washington University School of Medicine, 400 S. Kingshighway Blvd., St. Louis, MO 63110, U.S.A.
insert from the liver library were fully sequenced using the double-stranded dideoxy chain termination method [7]. Polylinker and unique internal restriction endonuclease sites and the Erase-a-Base (Sigma) nested deletion system were employed to subclone and sequence the larger clones. Sequence confirmation was achieved by using oligonucleotide primers to read the opposite strand, or by verification of multiple overlapping sequences produced by nested deletions. To confirm that the 4.2 kb transcript was fully processed and located in the cytoplasm, total cellular R N A isolated by acid guanidinium thiocyanate-phenol-chloroform extraction method [8] and total cytoplasmic R N A [9] were enriched for poly(A) + R N A by affinity chromatography on oligo(dT)-celhilose [10]. 10 /~g of total cellular or cytoplasmic RNA, or 2 ~g of poly(A) + RNA was size fractionated on a 1% agarose, 200 mM 4-morpholinepropanesulfonic acid (pH 7.4), 1 mM EDTA and 18% formaldehyde gel, transferred to nylon (Hybond, Amersham) and hybridized to 32p-labeled antisense R N A transcribed from Sp6 or T7 (Promega). The blot was washed and exposed to X A R film at - 7 0 o C. The entire nucleotide sequences for the unique MnSOD cDNAs are depicted in Fig. 1. Dramatic sequence differences are found in the 3'-untranslated sequences and the polyadenylation signals. The longest Mn-SOD c D N A (Mn-SOD 1) contains within its sequence, both of the 240 nucleotide 3' untranslated cassettes of the short Mn-SOD cDNAs 4 and 5. It neglects the AGTAAA polyadenylation signal [11] of Mn-SOD 4 (which is almost identical to the shorter Mn-SOD cDNAs described by Wispe [2] and Ho [1]) and utilizes
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
251 GATCTGGTCGACCTGGCTACCGGCTTCGGCAGCGGCTTCAG~AGATCGGCGGCATCAGCGGTAGCACCAGCACTAGCAGCATGTTGAGCCGGGCAGTG 98
MetLeuSerArgAlaWal TGCGG•A•CAGCAGGCAGCTGGCTCCGG•TTTGGGGTATCTGGGCTC•AGGCAGAAGCA•AG•CT•C•CGACCTGCCCTACGACTACGGCGCCCTGGAAC•T CysG~YThrSerArgG~nLeuA~aPr~A~aLeuG~yTyrLeuG1ySerArgG~nLYsHisSerLeuPr~AspLeu~r~TyrAspTyrG~yA~aLeuG1uPr~
200
CACATCAACGCGCAGATCATGCAGCTGCACCACAGCAAGCACCA~GCGGCCTACGTGAACAACCTGAACGTCACCGAGGAGAAGTACCAGGAGGCGTTGGCC HisIleAsnA•aG•nI•eMetG•nLeuHisHisSerLysHisHisA•aA•aTyrVa•AsnAsnLeuAsnVa•ThrG•uG•uLysTyrG•nG•uA•aLeuA•a
302
AAGGGAGATGTTACAGCCCAGATAG~TCTTCAG~CTG~ACTGAAGTT~AATGGTGGTGGTCATATCAATCATAGCATTTTCTGGACAAACCTCAGCCCTAAC LysG•yAspVa•ThrA•aG•nI•eA•aLeuG•nPr•A•aLeuLysPh•AsnG•yG•yG•yHisI••AsnHisS•rI•ePheTrpThrA•nLeuSerPr•Asn
404
GGTGGTGGAGAAC••AAAGGGGAGTTG•TGGAAGC•ATCAAACGTGA•TTTGGTT•CTTTGA•AAGTTTAAGGAGAAG•TGA•GG•TGCATCTGTTGGTGT• 506 G~yG~yG~YG~uPr~Ly~G~yG~uLeuLeuG~uA~aI~eLysArgAspPheG~yS~rPheAs~LysPheLysG~uLysLeuThrA~aA~aSerVa~G~yVa~ CAAGGCTCAGGTTGGGGTTGGCTTGGTTTCAATAAGGAACGGGGA•A•TTACAAATTGCTG•TTGT••AAAT•AGGAT••A•TG•AAGGAA•AA•AGGC•TT 608 G•nG•ySerG•yTrpG•yTrPLeuG•yPheAsnLysG•uArgG•yHisLeuG•nI•eA•aA•aCysPr•AsnG•nAspPr•L•uG•nG•yThrThrG•yLeu ATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTACTACCTTCAGTATAAAAATGTCAGGCCTGATTATCTAAAAGCTATTTGGAATGTAATCAACTGG I~ePr~LeuLeuG~yI~eAsPva~TrpG~uHi~A~aTyrTyrLeuG~nTyrLYsA~nVa~ArgPr~AspTyrLeuL~A~aI~eTr~AsnVa~I~eAsnTrp GAGAATGTAACTGAAAGATACATGGCTTGCAAAAAGTAA GluAsnValThrGluArgTyrMetAlaCysLysLys M~ SOD Mn SOD Mn SOD
1 ACCACGATCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAG 4 ACCACGATCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAG 5 A C C A C G A T C G T T A T G C T G A .......
1 TGGTATAGAGTACTGCAGAATACAGTAAGCTGCTCTATTGTAGCATTTCTTGATGTTGCTTAGTCACTTATTTCATAAACCAACTTAATGTTCTGAATCA
710
800
900
4 TGGTATAGAGTACTG•AGAATA•AGTAAG•TGCTCTATTGTAG•ATTTCTTGATGTTGCTTAGTCACTTATTTCATAAA•CAACTTAATGTT•TGAATCA I ATTTCTTA•TAAACATTTTGTTATTGGGCAAGTGATTGAAAATAGTAAATGCTTTGGTGGTGGATTGAAACTCTGAATTGGGACATTTTCTTCAGAGAGC 4 ATTTCTTACTAAACATTTTGTTATTGGGCAAGTGATTGAAAAAAAAAAAAAAAAAAAAAA TAAATTACAACTTTGTCATTTTATAAAACCATCAAAAATATT•CATCCATATATACTTTGGGGACTTGTAGGGATGCCTTTCTAGTCCTATTCTATTGCA
i000 ii00
GTTATAGAAAATCTAGT•TTTTG••••AGTTACTTAAAAATAAAATATTAACACTTTCCCAAGGGAAA•ACTCGGCTTTCTATAGAAAATTG•ACTTTTT 1200 GTCGAGTAATTCTGCAGTGATACTTCTGGTAGATGTCACCCAGTGGTTTTTGTTAGGTCAATGTTCCTGTATATAGTTTTTG•AAATAGAG•TGTATA•T 1300 GTTTAAATGTAGCAGGTGAACTGAACTGGGGTTTGCTCACCTGCACAGTAAAGGCAAACTTCAACAGCAAAACTGCAAAAAGGTGGTTTTTGCAGTAGGA GAAAGGAGGATGTTTATTTGCAGGGCG••AAGCAAGGAGAATTGGG•AGCTCATGCTTGAGACC•AATCTCCAGATGATGACCTACAAGCTAGGATATTT AAAGGCAGTGGTAAATTTCAGGAAAGCAGAAGTTAAAGGCAAAATTGTAAATCAGTCGAGACTGGGTGCCTTCAGGGTGGTATGGCTAGTATCA••AAAA TTGTAAATCACTACATGAAGCTTATATATTGGTTTGGCCTGA~AGGTGAAGTGGGTAGGCAGGGGGCGGGCTTACAGGTTATGGTGGATT~AAAGACTCC
CTGATTTGTGATTGGTTAAGGAAGCAAAGCTTTGTCTAAAAA•TTGGGTc•G•GAGAAGGAA•AATAGT•TG••AGC•CCTCAGGAAAGAA•A•TGAGAGC AAAGAATGGAGGTCAGAGTTAGTCCCTGGTGTTCCCCCTTATCTGACGTCTGTGTGAATCCCATTTGGTGGGGGTCTGGGTTTCTGAAAAGCTAGCTCAG GGGCA~GTGTTAAGGATGT~TCTAGGTGACTCTAACTT~CCTGGCTATTGTTTGAAACTGTTATGACCTTCTTGCTTATCAGCTTGCTGGTTTCCTTCTC GGGGCGAGCTGGGTGCCTGGAGTTTTCGGTGAAGGAAACTCAAGATTCTCCTTTATTTCTGTGCTTGTGGGAAT~cTGcA~cAAAGAGGGGTCCC TGACTAC~GT~TCACAGGGATACTTTTTGTATATTTGGCTTAGCATTCATACATTTTGGCCATGTTGGTTTCCATCCATCTGGCCTAATTTACTGTTTTT
GAATATTTCCATTTGTTT•TAAATGTAA•TAcAGATAATAG•ATGGGGTGAGCAA•ATCTGATGTA•ATAGGTTTATCTCCTATTGGAATATTTTCTTTA TATAGGCGTTTTTTTTTTTTCTTTTTTTTTGGAGACAGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCG•GACcGGAG•TcAcTG•AA••T•cA
~TT~C~GGGTT~AAGTGATTGT~CCACCTCAGCCTCCTGAATAG~TGGGATTATAGGTGCATGCTACCATGCCTGGCTACTT~TTGTATTTTTAGCAGAG ACAGGGTTTCACCATGTTGG•CAGGGTGGTCTCGAA•TCCTGAC•T•AAGTGAT••GT•TGGCTCAGCCTCCCAAATGCTGGGATTACAGGTGTGAGCCA CTGCACCTGGCCTATATAGGCTTTTTTCTTAAACCTATTTAGTAATGTTTTCCCAAGTTTATTTTTTATTTTTAATTTTTTTCCCCAAGTTTATTTTTCT ATTTTTTTTTCATGGAAAATGGGGTAACTTAGCAGTTTCAATATTGAAGACTGAAGTTTAAAAAAAAATTTAAATTCAAGGTACTTTTAAATTTCAGTTA GGAAAAGTAGGGCTTTAAA-~TTATTAGAGACAAGTACCAAAGCGGTGTGTGTATGTGTGTGTGTGTATGCATGCTTGTGGATTGGA~AAACTTTGGAGAC
TGATTACTTTTCATTATATATGTGTCACAGTGAAACAGCTTTTATGTGTCATGTAAGATTACTGCTTGCCTCTCTAAGGAAGGTCGTGACTGTTTAAATA GACGGGCAAGGTGGAA~TTTTGAAAGATGAGCTTTTGAATATAAGTTGTCTGCTAGATCATGGTTTGTATTGAACTAACAAGGTTTGCAGATCTGCTGA CTTATATAAGCTTTTTGATTCCTACTAAGCTTTAAGATTTAAAAAATGTTCAATGTTGAAATTTCTGTGGGGCTCTATTTTTG•TTTGGcTTT•TGGGTG
AGAGAGTGAGGAAGCATTCTTTCCTTCACTAAGTTTGT~TTTCTTGTCTTCTGGATAGATTGATTTTAAGAGACTAAGGGAATTTACAACTAGCGCTTTT AGTCCATCTGGTGGGAAAGGAGACTGTAGATTGTTAGGGCTGGG•GGGTGA•TCACAT•TGTAAT•CcCAGCACTTTGGGAGGCCAAGGCAGG•AGAACA CTTGAAGGAGTTCAAGACCAGCCGTGGCCCAACGTGGTGAAACCCTGTCTCTACTAAAAATACCAAGAAATTGTTTAGCTCTGTTTTTCATATAGAAAAT AGAAAAGGTAAATTCCGGTTTTT•TTCTGAAAAGAACAAGTATTGTTCATCCAAGAAGGGTTTTTGTGACTGAATCAGCAGTGCCTGCCCTAGTATAGTG
TGCTTCAAAAACCTCAGCATGATTAGTGTTGGAGCAAAACAAGGAAGCAAAGCAAATA•AGTTTTTGAAATTCTAT•TGTTG•TTGAA•TATTTTGTAAT AATTAAACTTTGATGTTGAGAAATCACAACTTTATTGTACACTTCATTGCAACTTGAAATTCATGGTCTTAAAAGTGAGATTGAATTTCTATTGAG~G~C
TTTAAAAAGTAATACCCAAA•CATAAAGGTTAAAATCTATGTATATTGAGTcATATCTAAACCACGGTATAAACATAAATTGTATTTCCTGTTTTAATTC 1 AGGGGAAGTACTGTTTGGGAAAGCTATTATTAGGTAAATGTTTTACAAATTACTGTTTCTCA•TTT•AGTCATAC••TAATGATCCCAGCAAGATAATGT Mn SOD
5
1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000
............ T C A T A C C C T A A T G A T C C C A G C A A G A T A A T G T
1 CCGTCTCTTCTAAGATGTCATCAAGCCTGGTACATACTAAAACCCTATAAGGTCCTGGATAATTTTTGTTTGATTATT•ATTGAAGAAA•ATTTATTTTC
4100
5 •CGT•T•TT•TAAGATGTCATCAAG•CTGGTACATACTAAAACCCTATAAGGTCCTGGATTAATTTTGTTTGATTATTCATTGAAGAAACATTTATTTTC 1 CAATTGTGTGGAAGTTTTTGACTGTTAATAAAAGAATCTGTCCAACCCAATCGGTAGGAGTCGACAGGATGAATTC 5 CAATTGTGTGGAAGTTTTTGACTGTTAATAAAAGAATCTGTCCAACCC
4176
Fig. 1. The DNA and derived proton sequence of human Mn-SOD cDNAs. The Y-untranslated re~ons of Mn-SOD 1, 4 and 5 ~e depicted. Coding re,on nucleoOde substitutions are denoted by astefieks and described in the text. The human Alu repetitive sequence element is indicated by underlimng. the A A T A A A polyadenylation signal of M n - S O D 5 (which completes a different shorter M n - S O D c D N A sequence reported by Beck [3]). M n - S O D 1 contains a consensus splice junction sequence with the invariant G T splice d o n o r and A G splice accepter sequences [12,13] at requisite positions to be able to produce
M n - S O D 5 f r o m M n - S O D 1. Bases 2659-2702 comprise an AT-rich sequence [ T T A T T T r [ T T A T ] T T T T A A T ] T T T . . . . [ T T A T T T T C T A T ] , similar to AU-rich sequences previously described in the Y-untranslated regions of several lymphokines, cytokines and protooncogenes m R N A s which are transiently expressed [14]
252 I
28S
2
3
4
-
region of this new Mn-SOD 1 cDNA and almost identical coding regions, including the mitochondrial leader sequences, raises the possibility of a regulatory role for Mn-SOD enzyme biosynthesis by the selection of particular Mn-SOD 3'-untranslated region cassettes.
18S -
Acknowledgements
Fig. 2. RNA blot analysis of total and cytoplasmic term placental RNA. Total (1) and cytoplasmic (2) RNA was prepared from term human placental RNA and poly(A)-enriched from total (3) and cytoplasmic (4) term placental R N A and separated by agarose gel electrophoresis. RNA was transferred to Nylon and hybridized to 32P-labeled antisense Mn-SOD 5 RNA. In both total and cytoplasmic human term placental RNA, there are two hybridizing bands, one at 4.2 kb and one at 1 kb. Poly(A) selection enriches the smaller Mn-SOD transcript. The large 4.2 kb Mn-SOD transcript is,found as a processed message abundantly in the cytoplasm.
and which have been implicated as mRNA-destabilizing sequences [15]. Mn-SOD 1 also contains, in its complementary strand, a sequence 84% homologous to an Alu repetitive element [16] (depicted by underlining) which is flanked by a 9 base repeat. As can be seen in Fig. 2, this large transcript is not heterologous nuclear unprocessed R N A (which can contain Alu repeats [16]) but exists abundantly in the cytoplasm as a processed R N A species. Additional sequence differences in these Mn-SOD cDNAs from the Mn-SOD sequences previously reported [1-4] include a C to T substitution at nucleotide 325 which results in a Thr to Ile amino acid substitution in all clones. Mn-SOD 4 contains an A to C substitution at base 159 which results in a Ser to Arg amino acid substitution and Mn-SOD 5 has a C to T substitution at base 198 causing a Pro to Leu substitution. Recognition of the co-existence of multiple human Mn-SOD transcripts, with dramatic sequence differences in the additional 3426 bases of 3'-untranslated
Many fruitful discussions and advice were provided by Drs. Harvey Colten, Arnold Strauss and James Grant. S.L. is a recipient of Clinical Investigator Award K8HD000885A from the National Institutes of Health. References I Ho, Y. and Crapo, J.D. (1988) FEBS 229, 256-260. 2 Wispe, J.R., Clark, J.C., Burhars, M.S., Kropp, K.E., Korfhagen, T.R. and Whitsett, J.A. (1989) Biochim. Biophys. Acta 994, 30-36. 3 Beck, Y., Oren, R., Amit, B., Levanon, A., Gorecki, M. and Hartman, J.R. (1987) Nucl. Acids Res. 15, 9076. 4 Heckl, K. (1988) Nucl. Acids Res. 16, 6224. 5 Xiang, K., Cox, N.J., Hallewell, R.A. and Bell, G.I. (1987) Nucl. Acids Res. 15, 7654. 6 Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 7 Sanger, F., Nickleu, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 8 Beyarsky, A., Vinogradova, T. and Rajewsky, K. (1989) Nucl. Acids Res. 17, 2920-2932. 9 Benoff, S. and Nadal-Ginard, B. (1979) Proc. Natl. Acad. Sci. USA 76, 1853-1857. 10 Davis, L.G., Dibner, M.D. and Battey, J.F. (1986) in Methods in Molecular Biology (Davis, L.G., Dibner, M.D. and Battey. J.F., eds), pp. 139-142, Elsevier, New York. 11 Birnsteil, M,L., Busslinger, M. and Strub, K. (1985) Cell 41, 349-359. 12 Cech, T.R. (1983) Cell 34, 713-716. 13 Sharp, P.A. (1981) Cell 23, 643-646, 14 Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S. and Cerami, A. (1985) Proc. Natl. Acad, Sci. USA 83, 1670-1674. 15 Shaw, G. and Kamen, R. (1986) Cell 46, 659-667. 16 Jelinek, W.R. (1982) Annu. Rev. Biochem. 51, 813-844.
