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Isolation and characterisation of an alternatively-spliced rat amelogenin cDNA: LRAP — a highly conserved, functional alternatively-spliced amelogenin?
BiocL et Bk
Biochimica et Biophysica Acta 1219 (199
Short Sequenceq
cterisation of an alter cDNA: LRAP - a highly conserved, conser ft amelo elogenin W.A. Bonass *, J. Kirkham, S.J. Brook Division of Oral Biology, Leeds Dental Institute, ln'~ Univ
C-spliced rat tl alternatively-s ;hore, C. Robinson LS2 9LU Leeds, UK
Received 20 May 19
tract ino-termini, but lacking the cc both the carboxy- and amino-tern i cDNA coding for a 59 amino acid polypeptide containing b~ sequence indicate The deduced polypeptide polypep 'at tooth enamel matrix protein, amelogenin, was cloned and sequenced. s me rat intra-exonic 3' splice acceptor a site. This alte alt, w a s derived from an amelogenin RNA molecule by using an alternative ans: the leucine-rich ar~ both cow and mouse enamel orgar uct is almost identical to products previously identified in bo~ product ha~ an important role in n.p). The conservation of this truncated polypeptide across the species suggests that it may have (LRAP). tooth enamel. eords: Amelogenin; cDNA; Alternative splicing; Sequence; (Rat) Keywords:
Tooth enamel is formed bby the deposition of relatively yapatite crystals in a proteinalarge, highly ordered hydroxyaF creted by the ameloblasts. The ceous extracellular matrix secreted ff the extracellular matrix are major protein components of the amelogenins. These are unique to enamel and are leveloping enamel crystals and thought to interact with the develo in so doing modulate crystal growth. Although there exists only one amelogenin gene in rodents [1] and two in humans [1,2] and the cow [3], amelogenin is known to exist in multiple forms in developing tooth enamel [4]. Some of this heterogeneity results from post-secretory modification of the primary translation product [5], however, it has also been shown that alternative splicing of the ibutes to this heterogeneity [6]. primary transcript also contributes A small polypeptide, namedd the leucine rich amelogenin tentified in developing bovine peptide (LRAP) has been identi or product of amelogenin gene enamel and represents a major expression [7]. Cloned cDNAA for LRAP has been isolated brary [6], and a similar cDNA from a bovine ameloblast librar, use ameloblast cDNA library, has been identified in a mouse
The sequence data reported in this manus the EMBL/GenBank Data Libraries under thc
Furthermore, a closely irelated polypeptide t developing porcine ena~ to be present in deve conserved alten appearance of such a hi ghly l number of species sug~ transcript between a nm aberation a unlikely to be a non-functional non-fu xmlecule in the formation c be an important molecu In the process of characterising ame clones from a rat enamel ename organ cDNA libra1 to a hum cDNA clone which hybridised h3 contained an ins probe, p87.4 [2] but which wh bp in length. Sequence analysis subsequentl this clone contained both bo the 5' and 3' ter the full length 790 bp rat amelogenin cDb 100% hom The truncated cDNA exhibits c of the full length cl corresponding regions have arisen from a sep that it is unlikely to hax full length amelogenin cDNA contains a c ~onding to a polypeptide of 589 bp correspondil acids. This includes 48 bp 1 which codes for i extracellul~ signal peptide which facilitates fa T h ~ t r H n p n t ~ c l r ' I ~)NA N I A rreported o here contains ion for a polypeptide of 7 IA corresponds to a signifi ogenin exon which codes ch domain which gives an
~A. Bonass et al. / Biochimica et Biophysica AAGAAATGGGGACCTGGATCTTGTTTGCCTGCCT M G T W I L F A C L 3 TACCACCTCATCCTGGGAGCCCTGGTTATATCAA L P P H P G S P G Y I N 5V6 TGAAGTGGTACCAGAGCATGATAAGGCAGCCGCC L K W Y Q S M I R Q P P
;TTTTGCTATG F A M 3T5 ~GGTGCTTACC V L T
58 17 118 37
CTCTGGAAGCTTGGCCAGCGACAGACAAGACCAA P L E A W P A T D K T K
?TCTTCCTGAG • L P E 6~7 ?GGATTAAAAA T D *
AGAAAATGAGAGAAC CGAAGTGGATACTTTGGTT
?AACTCAAGAA
298
299
C A C A A T G A T T T G T G C C T AT CA AC A T AA GGTAAATTCTG AC TA CC AT CTT
tAGTATCATTA
358
359
G C A G A T A A T A A A A T G T T T T A A A A A T~T C CAAA AAAAAAAAAAAA
I. Nucleotide sequence and deduced amino acid sequence of the rat LRAP i
eDNA
ced from reference [6]. The closed triangle indicatesthe position of of the th alternatiw
178 57 238
;tical arrowheads mark the intron e underlined amino acid residues
[de. The asterisk represents the termination codon.
1
2
3
4 5
6
7
1
2
3
4 5
6
7
Fig. 2. Mechanism for generating the alternatively spliced amelogenin The black bars indicate the exons that are spliced together. The numberin bering is that used in reference [5]. (a) In the major amelogenin transcri cript six exons are spliced together to produce a mature mRNA. Exoni 4 is rarely incorporated. (b) In the production of mRNA for LRAP exon 5 is spliced into an alternative acceptor site in exon 6, resulting in the loss of 121 amino acid codons.
(LRAP kP).
characteristic amino acid corn ,mposition. The sequence data suggest that one of the glutamine co mine codons (CAG) present in exon six acts as an internal splice ~plice acceptor site resulting in the loss of 363 coding nucleotides eotides (Fig. 2). In a previous study it was reported that the Ihe large exon coding for the proline and glutamine rich domain of amelogenin has arisen via multiple tandem duplications of a dodecanucleotide motif which includes les the codons for glutamine and proline [9]. It is of sig~ gnificant interest that the sequences missing from the truncated uncated eDNA, reported here, correspond to these putative tandem repeats, Following cleavage of the: signal peptide, the Rat LRAP homologue would comprisee a 59 amino acid mature
Rat
poly nificantly shorter than th leng lin which comprises a 11 poly e truncated polypeptide leuc! ; which has led to the poly fetal bovine enarr F U ~ y I J ~ F L = U ~ ~ u * a Ltted ~ u from uu the leucine rich amelo~,genin peptide (LRA ing this from the glutan [utamine-rich full length is clear from a compmrison of the full amelogenin and LRAP that these molecul significantly different structures s and there1 different properties. It could be argued that t this cDNA repr splicing aberation which whic has no function ii of tooth enamel, however, howe~ the isolation of clones from mouse and ant cow cDNA libral unlikely. Indeed LRAP has been identifiec bovine enamel and characterised ch by prot [10]. A comparison of the deduced amino of the mature truncated ancated amelogenin relatec 3) indicates that in eac each case the altemati used the same glutami lutamine codon as an all acceptor site. This extensive exte conservation alternatively spliced pre )roduct suggests that tl to represent an artifact and that the truncal may be essential for normal no development o The findings in this study confirm the alternatively spliced al amelogenin mRNA
M P L P P H P G S P G Y I N L S Y E V L T P L K W Y Q S M I R Q P P L S P I L P E L5PLEAWPATDKTI~EEVD PLEAWP * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *mr***************** ****
Mouse
M P L P P H P G S P G Y I N L S Y E V L T P L K W Y Q S M I R Q P P L S P I L P E L P LLEEAAWWPP A T D K T K R E E V D
Cow
M P L P P H P G H P G Y I N F S Y E V L T P L K W Y Q S M I R H P P L P P M L P D L P LPELAEWAPW P A T D K T K R E E V D
********
*****
****************
*** * ** *k************ * * * * * * * * * * *
~*************~*** Pig
Fig. 3. Alignment of primary amino acid se identical amino acid residues in adjacent seql
5PLEAWPATDKTKREEVD
ow and pig. The asterisks indica
KA. Bonass et al. / Biochimica et Biophysica
acisor. The alternatively at is identical to a major e [6] and almost identical w [7] and the pig [8]. In peptide lacking a major that several other alternaproduced; adding to the ecules found in the extraalar matrix of developing tooth enamel. Some of these :natively spliced products would be predicted by a lel based on the duplication of splice acceptor sites, lting in the presence of 18 CAG (glutamine) codons ted in exon six of the rat amelogenin [9]. "hese studies indicate a very high level of conservation le alternatively-spliced LRAP across the species implya specific role in enamel development. In addition, e studies indicate that the amelogenin transcriptional may be a useful model system in which to study the s of splice acceptor site selection. It is known that :native splicing of some mRNA molecules is developttally regulated [11]. The presence of at least 18 poten3' splice acceptor sites in a single exon in the amelogenin in gene may prompt further analysis of this gene for determinin rmining basic mechanisms of RNA splicing.
Refe [1] I [2] t
[3] < i
[4] t t [5] ~' q
mdas, T.K., Shapiro, L.J., Slavkil nomics 4, 162-168. akenaka, O. and Nakagome, Y. (1 lb, E., Herold, R., Risser, M., Dinj Termine, T. and Rosenbloom, J t079. and Moradian-Oldak, J. (1993) ] 197, 248-255. )renstein, S., Franzel, A. and N~ ! (Fearnhead, R.W., ed.), p. 286,
C
[6] I i t ] [7] ( q ( [8] ( [91 !
tmer, J.P., Bringas, Jr.,P., Hsu, M., Thiemann, F., Snead, M.L., (1992) Biochem. Biophys. Re
Golub, E., Ding, W., Shimokaw i Rosenbloom, J. (1991) Bioche 1306-1312. Tanabe, T., Fukae, M. and St ~t. 54, 69-75. (obinson, P.A,, Kirkham, J., Shor $ 3iochem. Biophys. Res. Commun Belcourt, A.B., Termine, J.D., [101 Fincham, A.G., Belcour Cothran, W.C. (1983) Bi Biochem. J. 211, 149-154 J.G. and Nadal-Ginard, B. [11] Smith, C.W.J., Patton, J.i Genet. 23, 527-577.
Biochimica et Biophysica Acta 1219 (199
Short Sequenceq
cterisation of an alter cDNA: LRAP - a highly conserved, conser ft amelo elogenin W.A. Bonass *, J. Kirkham, S.J. Brook Division of Oral Biology, Leeds Dental Institute, ln'~ Univ
C-spliced rat tl alternatively-s ;hore, C. Robinson LS2 9LU Leeds, UK
Received 20 May 19
tract ino-termini, but lacking the cc both the carboxy- and amino-tern i cDNA coding for a 59 amino acid polypeptide containing b~ sequence indicate The deduced polypeptide polypep 'at tooth enamel matrix protein, amelogenin, was cloned and sequenced. s me rat intra-exonic 3' splice acceptor a site. This alte alt, w a s derived from an amelogenin RNA molecule by using an alternative ans: the leucine-rich ar~ both cow and mouse enamel orgar uct is almost identical to products previously identified in bo~ product ha~ an important role in n.p). The conservation of this truncated polypeptide across the species suggests that it may have (LRAP). tooth enamel. eords: Amelogenin; cDNA; Alternative splicing; Sequence; (Rat) Keywords:
Tooth enamel is formed bby the deposition of relatively yapatite crystals in a proteinalarge, highly ordered hydroxyaF creted by the ameloblasts. The ceous extracellular matrix secreted ff the extracellular matrix are major protein components of the amelogenins. These are unique to enamel and are leveloping enamel crystals and thought to interact with the develo in so doing modulate crystal growth. Although there exists only one amelogenin gene in rodents [1] and two in humans [1,2] and the cow [3], amelogenin is known to exist in multiple forms in developing tooth enamel [4]. Some of this heterogeneity results from post-secretory modification of the primary translation product [5], however, it has also been shown that alternative splicing of the ibutes to this heterogeneity [6]. primary transcript also contributes A small polypeptide, namedd the leucine rich amelogenin tentified in developing bovine peptide (LRAP) has been identi or product of amelogenin gene enamel and represents a major expression [7]. Cloned cDNAA for LRAP has been isolated brary [6], and a similar cDNA from a bovine ameloblast librar, use ameloblast cDNA library, has been identified in a mouse
The sequence data reported in this manus the EMBL/GenBank Data Libraries under thc
Furthermore, a closely irelated polypeptide t developing porcine ena~ to be present in deve conserved alten appearance of such a hi ghly l number of species sug~ transcript between a nm aberation a unlikely to be a non-functional non-fu xmlecule in the formation c be an important molecu In the process of characterising ame clones from a rat enamel ename organ cDNA libra1 to a hum cDNA clone which hybridised h3 contained an ins probe, p87.4 [2] but which wh bp in length. Sequence analysis subsequentl this clone contained both bo the 5' and 3' ter the full length 790 bp rat amelogenin cDb 100% hom The truncated cDNA exhibits c of the full length cl corresponding regions have arisen from a sep that it is unlikely to hax full length amelogenin cDNA contains a c ~onding to a polypeptide of 589 bp correspondil acids. This includes 48 bp 1 which codes for i extracellul~ signal peptide which facilitates fa T h ~ t r H n p n t ~ c l r ' I ~)NA N I A rreported o here contains ion for a polypeptide of 7 IA corresponds to a signifi ogenin exon which codes ch domain which gives an
~A. Bonass et al. / Biochimica et Biophysica AAGAAATGGGGACCTGGATCTTGTTTGCCTGCCT M G T W I L F A C L 3 TACCACCTCATCCTGGGAGCCCTGGTTATATCAA L P P H P G S P G Y I N 5V6 TGAAGTGGTACCAGAGCATGATAAGGCAGCCGCC L K W Y Q S M I R Q P P
;TTTTGCTATG F A M 3T5 ~GGTGCTTACC V L T
58 17 118 37
CTCTGGAAGCTTGGCCAGCGACAGACAAGACCAA P L E A W P A T D K T K
?TCTTCCTGAG • L P E 6~7 ?GGATTAAAAA T D *
AGAAAATGAGAGAAC CGAAGTGGATACTTTGGTT
?AACTCAAGAA
298
299
C A C A A T G A T T T G T G C C T AT CA AC A T AA GGTAAATTCTG AC TA CC AT CTT
tAGTATCATTA
358
359
G C A G A T A A T A A A A T G T T T T A A A A A T~T C CAAA AAAAAAAAAAAA
I. Nucleotide sequence and deduced amino acid sequence of the rat LRAP i
eDNA
ced from reference [6]. The closed triangle indicatesthe position of of the th alternatiw
178 57 238
;tical arrowheads mark the intron e underlined amino acid residues
[de. The asterisk represents the termination codon.
1
2
3
4 5
6
7
1
2
3
4 5
6
7
Fig. 2. Mechanism for generating the alternatively spliced amelogenin The black bars indicate the exons that are spliced together. The numberin bering is that used in reference [5]. (a) In the major amelogenin transcri cript six exons are spliced together to produce a mature mRNA. Exoni 4 is rarely incorporated. (b) In the production of mRNA for LRAP exon 5 is spliced into an alternative acceptor site in exon 6, resulting in the loss of 121 amino acid codons.
(LRAP kP).
characteristic amino acid corn ,mposition. The sequence data suggest that one of the glutamine co mine codons (CAG) present in exon six acts as an internal splice ~plice acceptor site resulting in the loss of 363 coding nucleotides eotides (Fig. 2). In a previous study it was reported that the Ihe large exon coding for the proline and glutamine rich domain of amelogenin has arisen via multiple tandem duplications of a dodecanucleotide motif which includes les the codons for glutamine and proline [9]. It is of sig~ gnificant interest that the sequences missing from the truncated uncated eDNA, reported here, correspond to these putative tandem repeats, Following cleavage of the: signal peptide, the Rat LRAP homologue would comprisee a 59 amino acid mature
Rat
poly nificantly shorter than th leng lin which comprises a 11 poly e truncated polypeptide leuc! ; which has led to the poly fetal bovine enarr F U ~ y I J ~ F L = U ~ ~ u * a Ltted ~ u from uu the leucine rich amelo~,genin peptide (LRA ing this from the glutan [utamine-rich full length is clear from a compmrison of the full amelogenin and LRAP that these molecul significantly different structures s and there1 different properties. It could be argued that t this cDNA repr splicing aberation which whic has no function ii of tooth enamel, however, howe~ the isolation of clones from mouse and ant cow cDNA libral unlikely. Indeed LRAP has been identifiec bovine enamel and characterised ch by prot [10]. A comparison of the deduced amino of the mature truncated ancated amelogenin relatec 3) indicates that in eac each case the altemati used the same glutami lutamine codon as an all acceptor site. This extensive exte conservation alternatively spliced pre )roduct suggests that tl to represent an artifact and that the truncal may be essential for normal no development o The findings in this study confirm the alternatively spliced al amelogenin mRNA
M P L P P H P G S P G Y I N L S Y E V L T P L K W Y Q S M I R Q P P L S P I L P E L5PLEAWPATDKTI~EEVD PLEAWP * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *mr***************** ****
Mouse
M P L P P H P G S P G Y I N L S Y E V L T P L K W Y Q S M I R Q P P L S P I L P E L P LLEEAAWWPP A T D K T K R E E V D
Cow
M P L P P H P G H P G Y I N F S Y E V L T P L K W Y Q S M I R H P P L P P M L P D L P LPELAEWAPW P A T D K T K R E E V D
********
*****
****************
*** * ** *k************ * * * * * * * * * * *
~*************~*** Pig
Fig. 3. Alignment of primary amino acid se identical amino acid residues in adjacent seql
5PLEAWPATDKTKREEVD
ow and pig. The asterisks indica
KA. Bonass et al. / Biochimica et Biophysica
acisor. The alternatively at is identical to a major e [6] and almost identical w [7] and the pig [8]. In peptide lacking a major that several other alternaproduced; adding to the ecules found in the extraalar matrix of developing tooth enamel. Some of these :natively spliced products would be predicted by a lel based on the duplication of splice acceptor sites, lting in the presence of 18 CAG (glutamine) codons ted in exon six of the rat amelogenin [9]. "hese studies indicate a very high level of conservation le alternatively-spliced LRAP across the species implya specific role in enamel development. In addition, e studies indicate that the amelogenin transcriptional may be a useful model system in which to study the s of splice acceptor site selection. It is known that :native splicing of some mRNA molecules is developttally regulated [11]. The presence of at least 18 poten3' splice acceptor sites in a single exon in the amelogenin in gene may prompt further analysis of this gene for determinin rmining basic mechanisms of RNA splicing.
Refe [1] I [2] t
[3] < i
[4] t t [5] ~' q
mdas, T.K., Shapiro, L.J., Slavkil nomics 4, 162-168. akenaka, O. and Nakagome, Y. (1 lb, E., Herold, R., Risser, M., Dinj Termine, T. and Rosenbloom, J t079. and Moradian-Oldak, J. (1993) ] 197, 248-255. )renstein, S., Franzel, A. and N~ ! (Fearnhead, R.W., ed.), p. 286,
C
[6] I i t ] [7] ( q ( [8] ( [91 !
tmer, J.P., Bringas, Jr.,P., Hsu, M., Thiemann, F., Snead, M.L., (1992) Biochem. Biophys. Re
Golub, E., Ding, W., Shimokaw i Rosenbloom, J. (1991) Bioche 1306-1312. Tanabe, T., Fukae, M. and St ~t. 54, 69-75. (obinson, P.A,, Kirkham, J., Shor $ 3iochem. Biophys. Res. Commun Belcourt, A.B., Termine, J.D., [101 Fincham, A.G., Belcour Cothran, W.C. (1983) Bi Biochem. J. 211, 149-154 J.G. and Nadal-Ginard, B. [11] Smith, C.W.J., Patton, J.i Genet. 23, 527-577.