the journal of chemistry vol. 269, no. 42, issue of and u ... · 26560 a novel and major isoform of...

THE JOURNAL OF BIOIQGICAL 0 1994 by The American Society for

CHEMISTRY Biochemistry and Molecular Biology, h e .

Vol. 269, No. 42, Issue of October 21, pp. 26559-26567,1994 Printed in U.S.A.

A Novel and Major Isoform of Tyrosine Hydroxylase in Drosophila Is Generated by Alternative RNA Processing*

(Received for publication, April 15, 1994, and in revised form, July 21, 1994)

Serge Birman, Bruce Morgan$, Matthew Anzivino, and Jay Hirsh From the Department of Biology, University of Virginia, Charlottesville, Virginia 22903

We report that two isoforms of Drosophila tyrosine hydroxylase protein are encoded via alternatively spliced exons. The major isoform (Type 11) contains a novel acidic extension of 71 amino acids in the amino- terminal regulatory domain, which is likely to alter the regulatory properties of the tyrosine hydroxylase protein. The minor isoform (Type I) corresponds to the cDNA sequence reported previously (1). We also report the structure of the Drosophila tyrosine hydroxylase (DTH) gene and the diversity and tissue localization of its transcripts. At least three types of DTH mRNA are generated from a single primary transcript through alternative splicing and polyadenylation. Type I1 mRNA is the most abundant tyrosine hydroxylase transcript in Drosophila and is found predominantly in the hypoderm throughout all stages of development. Type I mRNA is present only in the CNS, where it is the primary form. The DTH transcripts detected in the CNS contain a longer 3’-untranslated region than the transcript expressed in the hypoderm, due to differential polyadenylation. In contrast, the same start site is used for DTH gene transcription in both tissues. These results show unexpected diversity in the DTH transcripts and point out possible mechanisms for differential regulation of tyrosine hydroxylase activity in the CNS and in the hypoderm.

Dopamine has two identified functions in insects: as a neurotransmitter in the central nervous system (CNS)’ (2) and as the precursor of quinones which are essential for pigmentation and hardening of the cuticle, i.e. the insect exoskeleton (3, 4). The enzymatic steps leading to biosynthesis of dopamine have been studied extensively in mammals (5). The first step is the oxidation of L-tyrosine to L-dopa (3,4-dihydroxy-~-phenylala- nine), catalyzed by the enzyme tyrosine hydroxylase (TH) (EC 1.14.16.21, in the presence of iron, oxygen, and a tetrahydro- biopterin cofactor. The subsequent decarboxylation of L-dopa to dopamine is efficiently catalyzed by the pyridoxal phosphate- dependent enzyme, L-aromatic amino acid decarboxylase. The

* This work was supported by a National Institutes of Health Grant GM27318 (to J. H.) and a research fellowship award from the Fogarty International Center (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. therefore be hereby marked “advertisement” in accordance with 18

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s) UI4395.

t Present address: Cutaneous Biology Research Center, Massachu- setts General Hospital, Charlestown, MA 02129. ’ The abbreviations used are: CNS, central nervous system; TH, ty-

rosine hydroxylase; DTH, Drosophila tyrosine hydroxylase; kb, kilo- base(s); PCR, polymerase chain reaction; RT, reverse transcription; MOPS, 3-(h”morpholino)propanesulfonic acid; RACE, rapid amplification of cDNA ends; bp, base pair(s); 3’-UTR, 3’-untranslated region; HTH, human tyrosine hydroxylase.

conversion of tyrosine to L-dopa is the rate-limiting step in this pathway (6).

Evidence supports a similar pathway for dopamine synthesis in the CNS of insects. The gene encoding Drosophila dopa decarboxylase (Ddc) , the homologue of L-aromatic amino acid decarboxylase, has been isolated (7) and characterized (8,9). This enzyme is required for decarboxylation of L-dopa and 5-hy- droxytryptophan, the respective precursors of dopamine and serotonin (10, 11). Immunocytochemical experiments demonstrate the presence of Ddc in the dopamine and serotonin neurons of the fly CNS (12-14). Likewise, a vertebrate TH antibody has been used to reveal tyrosine hydroxylase-like immunore- activity in catecholaminergic neurons in the CNS of insects (15-17). TH enzyme activity was demonstrated in fly head extracts (1). In addition, inclusion of tyrosine in the incubation medium of an isolated cerebral ganglion from cockroach stimu- lates L-dopa synthesis, but not in the presence of a tyrosine hydroxylase inhibitor (18). A single cDNA encoding Drosophila tyrosine hydroxylase (DTH) has been isolated from an adult head cDNA library with a rat TH probe (1).

The cuticle is secreted by the hypoderm cells at each molt during the life cycle of Drosophila (19). Several lines of evidence demonstrate that the biosynthesis of dopamine in Drosophila follows the same enzymatic pathway in the hypoderm as in the CNS. Peaks of dopa decarboxylase activity (20) and hypodermal dopamine synthesis (21) are synchronized with cuticle formation, whereas CNS Ddc activity remains relatively constant (22). Mutations in the Ddc locus (23,24), as well as in the pale locus, which most likely corresponds to D T H (25-27), result in unpigmented Drosophila embryos that are unable to hatch. Separable upstream DNA regulatory elements that control Drosophila Ddc expression in the CNS and in the hypoderm have been characterized (22,28-31), and the Ddc primary transcript is alternatively spliced in these two tissues (8, 9, 32). In contrast, nothing is known about the tissue-specific regulation of DTH gene expression.

The mammalian TH gene has been characterized in several species (33-38). Generally, the gene encodes a single form of the enzyme. An exception is found in higher primates, where alternative splicing of the TH primary transcript produces several isoenzymes: two isoforms in monkeys (39) and four in humans (40,411. Here we report the structure of the DTH gene and the diversity and tissue distribution of its transcripts. We find that at all stages of development, two forms of DTH mRNAs are produced through alternative splicing of a single DTH primary transcript, one of which predominates in the CNS and the other in the hypoderm. The major DTH mRNA encodes a novel tyrosine hydroxylase protein which is preferentially expressed in the hypoderm: the hypodermal DTH mRNA contains two additional contiguous coding exons which add an acidic domain of 71 amino acids in the regulatory part of the non-neural enzyme. In addition, the polyadenylation site recognized in the hypoderm is skipped in the CNS, such that the CNS DTH mRNAs are characterized by a longer 3’-untranslated region. The pos-

26559

26560 A Novel and Major Isoform of Drosophila Tyrosine Hydroxylase

sible significance of this alternative RNA processing in the regulation of tyrosine hydroxylase activity is discussed. A comparison of the exon structure of the Drosophila and human TH genes is also presented.

EXPERIMENTAL PROCEDURES Experiments have been performed with the Oregon R strain of Dro-

sophila melanogaster. Library Screening-The D. melanogaster pNB40 cDNA library from

12- to 24-h embryos (42) and A Charon 4 genomic library were gifts from N. H. Brown and T. Maniatis, respectively. DTH cDNA and genomic clones were isolated by homology screening with a fragment from a rat tyrosine hydroxylase cDNA (a kind gift from D. M. Chikaraishi) under low stringency conditions (43). About 30 positive cDNA clones and seven genomic A clones were obtained. The 4.5- and 6.7-kb EcoRI DNA fragments from the A clones that hybridized to the tyrosine hydroxylase cDNA were subcloned into pUC19.

DNA Sequencing-The cDNA and genomic clones were sequenced on both strands by the dideoxy method of Sanger et al. (44) in the presence of C I - ~ ~ S - ~ A T P (sequencing grade from DuPont NEN) following the guidelines of the Sequenase 2.0 kit (U. S. Biochemical Corp.). Polym- erase chain reaction (PCRhgenerated DNA segments were sequenced directly with the Circumvent kit from New England Biolabs, as suggested by the supplier, except that the length of the denaturation/ annealinglelongation steps were increased to 1 min. In addition, a chase step was included after each sequencing reaction using terminal de- oxynucleotidyltransferase (Boehringer Mannheim), as described in Ref. 52. Computer analysis of the sequences was performed on a Macintosh computer running DNA Strider (Christian Marck). Isoelectric points were estimated by iteratively solving the pK, equations of ionizable groups in protein sequences.

Primer Preparation-Oligodeoxynucleotides were synthesized with a Milligen Cyclone Plus apparatus. They were decoupled from the column beads by heating for 30 min a t 80 "C in 30% ammonia, 3.3% triethyl- amine. Primers were then cleaned by butanol precipitation and, if required, purified on polyacrylamide gels. They were resuspended a t approximately 10 nmoVml in TE (10 m~ Tris, pH 8.0, 1 m~ EDTA) and stored at 4 "C.

Polymerase Chain Reaction-The PCR medium was composed of 20 m~ MOPS, pH 8.1, 5 m~ MgCI,, 5 m~ sodium isocitrate, and 10 pg/ml gelatin., Amplifications were done in a final volume of 10 pl, in the presence of 0.4 m~ deoxynucleotides, 15 p~ of each primer, and 2.5 units of l hq DNA polymerase (Boehringer Mannheim). The reaction mixture was covered by a drop of paraffin oil. Care was taken to keep the tubes on ice until the heating block was at the denaturation temperature to minimize nonspecifically primed DNA synthesis. The thermal cycler used was a Coy model 60. Unless specified otherwise, 1 min at 94 "C, 1 min a t 65 "C, and 40 s at 72 "C cycles were repeated 35 times, followed by a 7 min a t 72 "C final step. PCR products were analyzed on 1% Seakem + 1.2% Nusieve (FMC Bioproducts) agarose minigels stained with 0.025% ethidium bromide.

For PCR amplification from the total cDNA library, 1-1.11 aliquots from a suspension of Escherichia coli transformed with the cDNA-containing plasmids was mixed with the PCR buffer and boiled for 10 min. After cooling on ice, primers, dNTPs, and Taq polymerase were added and the PCR reaction started.

RNA Extraction--Total RNA was extracted from Drosophila using an adaptation of the method of Chomczynski and Sacchi (45). Tissues were dissected on an ice-cold block in calcium-free Drosophila Ringer's solution. Samples (typically 2 adult flies or 15 adult heads or 30 third instar larval CNS) were transferred into microcentrifuge tubes and immediately homogenized in 150 pl of guanidine isothiocyanate-containing Solution D (45). After addition of 15 pl of 2 M NaOAc, pH 4, 150 pl of water-saturated phenol and 30 pl of Sevag (chloroform-isoamyl alcohol, 24:l (v/v)), tubes were vortexed, kept on ice for 30 min, and centrifuged for 15 min at 4 "C. Only 120 pl of the aqueous phase was carefully pipetted out to avoid disturbing the interface that contains the DNA. The aqueous phase was re-extracted once with 30 pl of water-saturated phenol + 30 pl of Sevag and once with 30 pl of Sevag. One volume of isopropyl alcohol was added and the RNA precipitated for 45 min at -70 "C. Pellets were resuspended in 30 p1 of TE, pH 7.5, and precipitated again with ethanol in the presence of 0.4 M LiCI. RNA was resuspended in 11 pl of TE immediately before the reverse transcription experiment.

S. Tabor, personal communication.

Reverse Dunscription-linked Polymerase Chain Reaction (RT- P C R k 9 pl of RNA solution and 2 pl(20 pmol) of a tyrosine hydroxylase antisense oligodeoxynucleotide were mixed and heated for 2 min at 68 "C. ARer cooling on ice, reverse transcription was performed in 50 mM Tris-HC1, pH 8.3 (at 37 "C), 75 m~ KC], 3 m~ MgCI, in a final volume of 20 pl, in the presence of 20 units of RNase Inhibitor (Boeh- ringer Mannheim) and 100 units of Superscript reverse transcriptase (Life Technologies, Inc.). Tubes were incubated for 75 min at 37 "C, and the reaction mixture was then diluted with 20 pl of TE, pH 8.0. 1-pl aliquots were used for PCR as described above. The oligodeoxynucleotides used for RT-PCR were (see Fig. 2): OTHl (TH antisense, exon E), 5'GGTATGGTGAGTTAACGTGG; OTH2 (TH antisense, exon E), 5'CAACAAAATCTCGTCCTCGGTGAGACC; OTH3 (TH sense, exon A), 5'GCCCAAAAGAACCGCGAGATGTTCGCCATC@ OTH4 (TH antisense, exon G), 5'TCTTGATCCCAATGl"'AC.

We determined that these RT-PCR amplification conditions yield a valid ratio of the two DTH mRNAs by the following criteria. First, analysis of the products obtained after various number of PCR cycles showed that the ratio of Type I to Type I1 DTH cDNAs did not change in the course of amplification, and second, amplification with two independent pairs of primers gave the same ratio between the two bands (data not shown). Third, when used as template in the same conditions, the pure cDNA clones corresponding to the two mRNA subtypes yielded similar amounts of PCR amplification products (Fig. l), demonstrating directly that the two cDNAs are equally good templates. Fourth, quan- titation of the ratio of the two bands on a Northern blot (Fig. 3) agrees with the ratio determined from the RT-PCR experiments.

Northern RNA Analysis-The method described in Ref. 5 was scaled up to extract RNA from about 200 adult heads. Poly(A)+ RNA was purified using the Poly(A)Tract kit from Promega. After agarose gel electrophoresis in denaturing conditions, RNA was transferred onto Nylon membranes and covalently coupled to the filter by W cross- linking. Prehybridizations and hybridizations were done at 42 "C (respectively for 4 and 16 h) in 50% (v/v) deionized formamide, 5 x SSC, 50 mM Na/Na, phosphate pH 6.8, 2.5 x Denhardt's solution, 0.1 mg/ml sheared salmon sperm DNA, 0.1% (wh) N-lauroylsarcosine and 0.02% (w/v) SDS. The filters were washed three times for 5 min a t 42 "C in 2 x SSC, 0.1% (w/v) SDS, followed by three times for 15 min at 60 "C in 0.2 x SSC, 0.1% (w/v) SDS. Exposure time was approximately 2 days. Between each hybridization, the blot was stripped of probe by boiling for 1 h in 0.01 x SSC, 1 m~ EDTA, 0.1% (w/v) SDS and checked for the absence of any residual radioactivity. The sizes of the RNAs detected were estimated by comparison with the 0.24-9.5-kb RNA ladder from Life Technologies, Inc.

Starting from PCR-amplified and purified TH cDNA fragments as template (about 10 ng), single-stranded radioactive probes were synthesized by thermal cycling in the presence of the antisense primer alone (32). Conditions were essentially as described above for PCR except that 50 pCi of [ C I - ~ ~ P I ~ C T P (3000 Ci/mmol, DuPont NEN) re- placed cold dCTP, and the cycling program was modified: 94 "C for 1 min, 55 "C for 3 min, 72 "C for 2 min.

Three different probes were used (represented in Fig. 6A): 1) probe "I + 11": bases 470-664 of DTH cDNA Type I, hybridizing to both forms of TH mRNA; 2) probe "11": bases 633-851 of DTH cDNA Type 11, hybridizing to exon C and D and thus specific for mRNA Type 11; 3) probe "3'": bases 5525-5822 of the TH primary transcript, hybridizing between the first two poly(A) signals in the distal part of the gene.

mRNA End Analysis--To map the 5' ends of DTH mRNAs, we used a PCR method formally equivalent to primer extension, by analyzing the 5' ends of the DTH cDNAs present in a unidirectional Drosophila embryo cDNA library. This library gives full-length clones in a large fraction of cases (42). An oligodeoxynucleotide was synthesized (ONB5', 5'GGTGACACTATAGAATACATTGC) that anneals to the pNB40 vector close to the 5' end of the cDNA insert. Two antisense tyrosine hydroxylase primers were alternatively used: OTH5 (B'TCCGACT- GCTCCTGTTCAAC GCTCTC), which hybridizes to exon D and is specific for DTH mRNA Q p e 11, and OTH6 (5'GAGACCGTAATCA"TGC- CTTGC), which hybridizes to the exon B-E junction and is specific for DTH mRNA Type 1. After heat denaturation of the cDNAlibrary (see 3) above), 15 cycles of asymmetric PCR (1 min at 94 "C, 3 min at 65 "C, 2 min a t 72 "C) were first performed in the presence of one TH primer to increase the relative intensity of the specific signal. Then, the generic primer ONB5' was added followed by 30 cycles of double-stranded PCR (1 min a t 94 "C, 1 min at 65 "C, 40 s at 72 "C).

The method of rapid amplification of cDNA ends (RACE-PCR) (46) was used to analyze the 3' end of the tyrosine hydroxylase mRNAs. Briefly, reverse transcription of adult Drosophila head RNAs was performed after annealing with a 49-mer gel-purified oligodeoxynucleotide

A Novel and Major Isoform of Drosophila Tyrosine Hydroxylase 26561

mRNA cDNA

Larva Adult Libr I1 I ”

1 2 3 4 5 6 7 FIG. 1. Drosophila TH mRNA species detected by RT-PCR

Lanes 1 4 , total RNA was extracted from third instar larvae or adults. TH mRNAs were converted into cDNA by reverse transcription with the primer OTHl (see Fig. 2 and “Experimental Procedures”). Reverse transcriptase was omitted in the negative controls (lanes 1 and 3) . Two other primers (OTH2 and OTH3) were used during PCR to amplify TH cDNA fragments. Lanes 5 and 6, same PCR amplifications starting from a 12-24-h embryonic cDNA library (lane 5, about 1 ng of total DNA) or isolated TH cDNAs clones (lanes 6 and 7,2.5 pg of each). The two types of TH cDNAs are indicated at the right. The lengths of the DNA fragments were confirmed by sequencing.

containing a specific sequence (here the phage T7 RNA polymerase promoter) fused to a 17-mer oligo(dT) stretch: 5”ACGGAATTCTAAT- A C G A C T C A C T A T A G G G A G A P . A primer adapter containing only the specific sequence (5’-ACGGAATTCTAATAC- GACTCACTATAGG), and a tyrosine hydroxylase sense primer (not shown) were used for PCR. Products from this first PCR were diluted 100-fold, then 1 1.11 from this dilution was re-amplified using the same primer adapter, but another more downstream sense tyrosine hydroxylase oligodeoxynucleotide. After gel separation, the bands obtained were purified and directly sequenced.

To confirm that the 5’ or 3’ ends thus obtained were not artifactual, sequences of the amplified fragments were determined and compared with a DTH genomic clone. In addition, the length of the DTH mRNAs deduced from these data are in agreement with the size estimated on Northern blot.

RESULTS

Two Tyrosine Hydroxylase mRNAs Are Generated by Alternative Splicing in Drosophila

To study the structure of the DTH gene transcripts, we utilized RT-PCR, a very sensitive method to detect mRNAs of low abundance. The primers used are described under “Experimen- tal Procedures” and represented in Fig. 2. Total RNA was extracted from third instar larvae or adult flies and annealed to the primer OTH1. After cDNA synthesis, a DNA fragment corresponding to the TH transcript was selectively amplified by PCR in the presence of the primers OTH2 and OTH3. We expected a 195-bp DNA fragment according to the published sequence of a single TH cDNA (1). However, the major product of the reverse transcription PCR is a fragment of 409 bp (Fig. 1, lanes 2 and 41, which is not detected in controls lacking reverse transcriptase (lanes 1 and 3) . The expected 195-bp amplification product is of lower abundance: this band is present in adults (lane 4 ) and, although not visible in lane 2, can also be detected in third instar larvae with additional rounds of PCR amplification (not shown). To confirm that the 409-bp band originates from a TH RNA, an independent pair of primers was used for PCR with similar results (not shown). We refer to this novel TH transcript as DTH mRNA Type 11.

Starting from a 12-24-h embryonic cDNA library, identical fragments were amplified by PCR with the same primers (OTH2 and OTH3), and no other fragments were detected (Fig. 1, lane 5). The 195-bp fragment is relatively more abundant at this early stage. Several TH cDNA clones were isolated by

screening the embryonic library with a rat TH probe, most of which corresponded to DTH mRNAType 11, as demonstrated by PCR (Fig. 1, lane 6). PCR amplification from a clone corresponding to DTH mRNAType I is also shown (Fig. 1, lune 7). In both cases, the same amount (2.5 pg) of pure plasmid DNA was used as PCR template, resulting in comparable levels of amplification for the two fragments (lanes 6 and 7). Therefore, the relatively lower amplification observed for the 195-bp fragment in lanes 2, 4, and 5 is most likely due to a lower abundance of DTH mRNA Type I (see also “Experimental Procedures”). These results show that at least two forms of TH mRNA are produced in Drosophila and that the previously characterized TH cDNA corresponds to a minor form.

To learn how the two mRNAs differ, the TH cDNA clones were sequenced. This revealed that DTH mRNA Type I1 contains an insertion of 213 bases in the translated region relative to mRNA Type I. This new fragment potentially encodes a segment of 71 amino acids. Isolation and sequencing of a genomic clone containing the TH gene showed that the difference between mRNA Type I and Type I1 originates from an alternative splicing of the primary transcript (Fig. 2). DTH mRNA type I1 contains two contiguous but distinct coding exons (C and D) that are skipped in DTH mRNA Type I. We have not detected a tyrosine hydroxylase transcript containing either exon C or exon D alone. Apparently, both exons are included or excluded as a pair when the primary transcript is spliced.

Fig. 2 also shows the intron-exon organization of the DTH gene. The primary transcript initially contains seven exons, all of which include some protein coding information. The 5’ and 3‘ ends of the gene were defined by further experiments described below. Using RT-PCR and primer pairs spanning all other introns, no other alternative splicing events were detected for the DTH gene (not shown). Type I and I1 thus appear to be the only forms of tyrosine hydroxylase mRNA generated by alternative splicing in D. melanogaster.

Drosophila TH mRNAs Also Differ in the Length of Their 3’4Jntranslated Regions

Fig. 2 shows the three potential polyadenylation signals (underlined) found on the TH gene downstream from the TAA stop codon. The first signal is separated by about 600 bp from the second one, whereas the third polyadenylation signal is 110 bp from the second. All the TH cDNAinserts we analyzed ended at bp 5268, 200 bases before the 5’-most polyadenylation site, probably due to an artifactual priming of the oligo(dT) primer a t a genomic poly(A) sequence during library construction. Therefore, the 3”untranslated region (3‘-UTR) of these clones is probably incomplete.

To analyze more precisely the 3’ end of the TH mRNAs, we used the method of RACE-PCR (46) (see “Experimental Proce- dures”). Two TH 3‘ end cDNA fragments of different length were amplified (data not shown). The length of these fragments suggested that only the first and the last polyadenylation signals of TH are used in vivo. This results was confirmed by sequencing the RACE-PCR products. The precise site of poly(A) addition is in both cases 21-23 bases after the polyadenylation signal (Fig. 2). Consequently, in addition to the TH mRNA dimorphism generated by alternative splicing (Types I and 111, TH mRNAs also differ by the length of the 3“untranslated region: the L Type (long 3‘-UTR) has an additional segment of 680 bases at its 3‘ end relative to the S Type (short 3’-UTR).

Assuming that splicing of the TH primary transcript is not coupled to choice of the polyadenylation site, four mRNA subtypes are theoretically possible: Types Is, I,, 11,, and 11,. To determine which of these exist in vivo, the lengths of the complete TH mRNAs were estimated by Northern analysis of

26562 A Novel and Major Isoform of Drosophila Tyrosine Hydroxylase I 10 I 20 I 30 I 40 I 50 I 60 I 70 I 80 I 90 I 100

..... tqaaqcaa acaatcqtcg qcaatcqqcc qaaacgttcq ttaacttqqc cqtcqqaatc gqaatctqqq qd 1 GCATGCATTG ATTCGCTATC ATTCGCCCTA AAGACTTG'IY: CCCAGCCTCA AGTAGTTTGC TGACTCTGTT TGTTGTAATA CAATTTAGCT GACAAGGATA

TTCTTGTCCA GCTCAATTCT AGACGCAAAC AGTTCAGTAC CAGGAGCGAT TTTCTTGAAA AACAAAACCA CCCAGCCAGA TCGCCAATAA GCAGTGMG 201 ATACACATCA CCACATCAGT TTCGAGTTCG CGAAAGGATC CAGCTAAAGT TCAACCGATC AACCATCTAT ATATCCCGCC G"CGA GAGTCGAGAG 101

tatttaab cqqcqgcaac tcqacataaa

A 301lTGTCAACACA TTTCGAGTGC TTATTGCAAA TCAAACAAGA AAAGCGTAAC CAAGCACATA AGCAAATAGT ATTCTAAGTG T?TTTGcCAA CACTTTCPGT1 ~ ~ ~. ~~ ~.~ .. 401

gAAATCCTA CAGTATTGAG gtgagtcgaa aatccagcaa ..... 501

AAATTTTTGT TGTTCACCCC AAACGAAAAC AACCCAAACA CACAAAACAA AATGATGGCC GTTGCAGCAG CCCAAAAGAA CCGCGAGATG TTCGCCATCA M M A V A A A O K N R E M F A I K

~ ~~~~~~ .~

OTH3 .. ... ctcttcg 1500

K S Y S I E 1501 cttttgtttc ca$VfRXCT ATCCATCCCG CCGTCGCAGC CTGGTGGATG ATGCCCGTTT CGAGACCCTG G'EGTCAAGC AGACCAAACA AACCGTCCTC

B 16OllGAGGAGGCCC GCAGCAAGGC AAAVftaag tgaatagggt ccaac.. . . . N G Y P S I R R R S I L V D D A R F E T L V V K Q T K Q T V L

E E A R S K A N """"- . . . . .ttat ttttatgatt ttaaa4ACGA TTC~CTGGAA GATTGCATIY: TGCAGGCTCA GGAGCACATTJI~OO

-1 100 200 300 400 500

1901 CCCTCCGAGC AAGATGTGGA GCTCCAGGAC GAGCATGCCA ATCTGGAGAA CCTGCCCTTG GAGGAATATG TTCCA tta atatccattt ccattt .... -----------o D S L E D C 1 V 0 A 0 E H 1 I

C I """""""- P S E Q D V E L Q D E H A N L E N L P L E E Y V X

D 2101 a E f f i A G GATGTTGAGT TTGAGAGCGT TGAACAGGAG CAGTCGGAGT CGCAGTCGCA GGAGCCAGAA GGCAAC V E E D Y E F E L L E O E O S "

2201 cagggggttc tagaa.. ... . . . . . t ttttttggcc Ctttt

O T H ~ 2601'AGATmGTT GGCCAATGCC GCCTCCGAAT CCTCGGATGC CGAGGCTGCC ATGCAGAGTG CCGCTTTGGT GGTCCGCCTC AAGGAGGGCA TCTCCTCCTT

2900 AAGTTGGACA TGACCCGTGG CAATCTGCTG CAGCTGATCC GCTCCCTCAG GCAGTCGGGC TCCTTCAGCA GCATGAATCT GATGGCCGAC AATAACTTGA 2801

2800 GGGTCGCATC CTCAAGGCCA TCGAAACCTT CCACGGCACC GTCCAGCATG TGGAGTCCCG TCAGTCGCGC GTGGAGGGCG TGGACCACGA ETCCTCATC 2701

2700

G R I L K A I E T F H G T V Q H V E S R Q S R V E G V D H D V L I

2901 ATGTCAAGGC TCCGTGGTTC CCCAAGCACG CCTCCGAATT GGATAACTGC AACCATCTGA TGACCAAGTA CGAGCCCGAT TTGGACATGA ACCACCCCGG 3000 K L D M T R G N L L Q L I S R L R Q S G S F S S M N L M A D N N L N

V K A P W F P K H A S E L D N C N H L M T K Y E P D L D M N H P G E 3001 ATTCGCCGAC AAGGTATACC GCCAGCGTCG CAAGGAAATT GCCGAGATCG CATTCGCCTA CAAGTACGGA GACCCGATCC CATTCATCGA CTACTCCGAT 3100

I L L A N A A S E S S D A E A A M Q S A A L V V R L K E G 1 S S L

F A D K V Y R Q R R K E I A E I A F A Y K Y G D P I P F I D Y S D 3101 3200 GTGGAGGTCA AGACCTGGCG CTCGGTGTTC AAGACCGTTC AGGATCTGGC TCCCAAGCAC GCCTGTGCCG AGTACCGGGC CGCCTTCCAG AAGCTCCAGG

V E V K T W R S V F K T V Q D L A P K H A C A E Y R A A F Q K L Q D 3201 3300 ATGAGCAGAT CTTCGTGGAG ACCCGTCTGC CCCAGTTGCA GGAGATGTCC GACTTTCTGC GCAAGAACAC CGGATTCTCT CTCCGTCCTG CCGCCGGTCT

E Q I F V E T R L P Q L Q E M S D F L R K N T G F S L R P A A G L

OTH1 I L T A R D F L A s L A F R I F o s T o y v R 3 v N s P Y H T P E P $ 3301 TTTGACTGCC CGGGACTTCC TTGCCTCCTT GGCCTTCCGC ATCTTCCAGA GCACCCAGTA TGTGC AC GTTAACTCAC CATACCACAC CCCCGAGC 3400

3401 taaqtatcta tcatcatat. .... . . . . .attattcta tqccattaca 3700 TCCAT TCACGAGCTG C"CACA TGCCCCTGCT GGCCGATCCC AGCTTCGCCC AGTTCTCGCA GGAGATTGGA CTGGCCTCGC TCGGTGCCTC

D S I H E L L G H M P L L A D P S F A Q F S Q E I G L A S L G A S GAAGAA ATCGAGAAGC mrccAc4gt gagttgattt tctaattg.. . . .

3401 taaqtatcta tcatcatat. .... ~ ~ ~ ~~ ~~~

3701 CkGACTCCAT TCACGAGCTG CTGGGTCACA TGCCCCTGCT GGCCGATCCC AGCTTCGCCC ACTTCTCGCA CGAGATTGGA CTCmCTCGC TCXXTCXTT~~~OO . . . . .attattcta tqccattaca 3700

Laattg.. . . . D E E I E K L S T 1

. . . ..tta ccattccatt tcttcakTA TACTGGTTCA CTGTTGAGM CGGTCTCTGCi4200

4201)AAGZAACATG GTCAGATCAA GGCCTACGGT GCTGGACTCC TGAGCTCCTA CGGTGAGCTG CTCCATGCCA TCAGCGACAA GTGCGAGCAC CGCGCCTTCG 4300 Y W F T V E F " " ' 1

K E H G Q I K A Y G A G L L S S Y G E L L H A I S D K C E H R A F E 4301 4400 AGCCCGCATC CACCGCCGTG CAGCCCTACC AGGATCAGGA GTACCAGCCC ATCTACTATG TGGCCGAGAG CTTCGAGGAT GCCAAGGACA AGTTCCGTCG

P A S T A V Q P Y Q D Q E Y Q P I Y Y V A E S F E D A K D K F R R 4401

4600 CACCAGATGA ACACGGAGAT TTTGCATCTG ACCAACGCCA TCTCCAAGTT GCGACGCCCG TITTAAGTGG ATGGGGGGGA GATATATGTA TGATATATAG 4501 W V S T M S R P F E V R F N P H T E R V E V L D S V D K L E T L V

4500 CTGGGTGAGC ACCATGTCGC GTCCATTCGA GGTGCGTTTC AACCCGCACA CCGAGCGCGT CGAGG'IY%TG GACTCCGTGG ACAAGCTGGA GACTCTGGTG

H Q M N T E I L H L T N A I S K L R R P F 4601

5400 TGGAAAACAA AATAGAATTT TCTTACATTG TACAATAACT GTAAACGACA ATATTACTAT TGTGTACTAT TATTATTATT ATCTATTATT ATACTTATGA 5301 5300 AGAGAGTATT TCTAAACAAA AATGATATAG ATACAACAAT CGAGCAAGGA ACAAATGGAA AACAAACAAA AAAAAAAAAC GG-GATA AAAAATGAAA 5201 5200 GTTITTGTAT TATTATTATC TTf2A"RT.X CTCTCACAAT ATTTACCTAG ACTATATAGA CACGCGATAT AGTATTCCCG GTATAGGAAG CTTCATTTAG 5101 5100 ATTAAG- GTGAAAAGCT GGGAAAGCAA AGTTCAGATA CATCCGCACT TAGTAGGCAA AGTACCACAC AAAAACCGAT TGTATACTCA GACAAAAATT 5001 5000 CCCAACCTGC ATATTTATCA CCTATTATTG GACCAAAAAA TCTTTAGACG TTTAAGTGAT GTGAAAACAA AGCCAAGCCA AGCCAAGCAA AGCAAAGCGA 4901 4900 TTTATTTAAT TGTCATCGAA AAGTTGTAAT TGTCGAATCG CGCACATGGT AAAAAACACA AAAATCAAGC ATA'MTA'MT GTTGATCACC CGATACACCA 4801 4800 ATTGACATAC CGCACAATCC CTCGTACACG TAATCCTTAA ACCAAAATTA AGGCATCGCG CATTATITTT TAlTTTTTGT TTATTG'MTT TpGmAATT 4701 4700 AAGCACCCAA AAACATCGCC CAAAAAACAC ACCAGCCACC CCTACCACCC AAACATACAC ATACAGGTGC AACGGTCTCA TTTATACAAA TTAATTTTAA

TTAAAATTAA TGAGTAGAAT CGAGGAATAT GATAAATATA TTTATGGGTG AGAAGAAGTG AATCTCATGT GAACTAAAAT CGAGCTATGT AGAAAATATT 5700 TCACAAATTT GATGAAATTA TATAAAATAA CGAAAAACCT TGGACCAATA CATATGTATT ATTATAAAGT AATTAAAATC AAAATATTTC AAAGTAAACA 5800 AAG CA TTGGGATCAA GAATATTACA TATTTATAGT TAACGTAGCA TGGAATTTTA TTGTTATTGT TCGATGTATT GTAGTPGCAA TTGGATCGAA 5900 A G C G A A GTGTTAACAT TTCTGCAAGC AATTAGCCAA ACTAATTACT AATTATATTC GCACATAAAT CTATATATAT AATCAACTTA AAGCGCAGTT 6000 I GGTCCCCAAG CCAGGCTTCG TTGGTTCTGA AAAATTCAAT AAAATTATAT GTTTGAATGG AGCACAAAAC GTGCAGATAT CGTAAATATA TATATAAATA16100 TATATATATA CACATTCTTC TCTATATGTA CA'MTAATCG CTATTATATG AATAAAGTTG AAATCGWTA AACTTG4aa cgaactaaaa ttctgggagg 6200

6201 ggctgtggca cgcccccaca attttacaat gattttttta ttttttattt gcacaccttg acttgccaga gatcc ..... I 10 I 20 I 30 1 40 I 50 I 60 1 7 0 I 80 I 90 I 100

FIG. 2. Sequence and intron-exon boundaries of the Drosophila TH gene. The TH gene was isolated by screening a D. melanogaster genomic library with a rat TH cDNA probe. Intron-exon structure was deduced by comparing the sequence of the genomic and cDNA clones. Exons

Dashed boxes indicate the alternatively spliced and polyadenylated exons. The complete DTH gene sequence, including introns, has been submitted are boxed (uppercase letters) and identified by capital letters A through G at the left. The protein sequence is shown below the translated portions.

to GenBank (accession number U14395). Apossible TATA box located 25 bp upstream from the start site of transcription is boxed, and three poly(A) signals in the 3'-UTR are underlined. The sites of polyadenylation used in vivo have been determined by RACE-PCR. Arrows indicate position of the primers used for RT-PCR (OTHl-OTH4). A consensus site for CAMP-dependent protein kinase (Ser32 in exon B) is also boxed. A cDNA sequence corresponding to the protein coding portions of exons A, B, E, F, and G has been reported previously (1).

poly(A)+ RNA from adult fly heads. Three radioactive TH scribed under "Experimental Procedures" and represented in probes of 200-300 bases in length were synthesized and hy- Fig. 6A. They hybridized, respectively (Fig. 3): (i) to a region of bridized successively to the same blot. These probes are de- 200 bp encompassing the exon B-E boundary in DTH mRNA

A Novel and Major Isoform of Drosophila Drosine Hydroxylase 26563

Probe: I + II II 3'

- 9.5 - 7.5

- 4.4 3.7 - 3.2 -

- 2.4

- 1.3

1 2 3 FIG. 3. Northern blot analysis of Drosophila TH mRNA species.

Poly(A)+ RNA was prepared from adult heads. A single blot was hybridized sequentially with probe I + I1 that recognizes both DTH mRNA Types I and I1 (lune 1 ), probe 11, specific for mRNAType I1 (lune 2), and finally probe 3' that hybridizes to the distal part of the 3'-UTR (lune 3 ) (see 'Experimental Procedures" and Fig. 6). No additional RNAs were detected after a longer exposure (not shown). RNA size markers are indicated at the right.

Type I (probe "Z + ZP, lane 11, (ii) to exons C and D in DTH mRNA Type I1 (probe "ZP, lane 2 1, and (iii) to the distal part of the 3"untranslated region (probe "3"', lane 3) .

The probe I + I1 was expected to hybridize to both alternatively spliced TH mRNAs, because it recognizes sequences common to Type I and Type 11. When this probe was used (Fig. 3, lane 11, a prominent 3.2-kb and a faint 3.7-kb band appeared. A similar Northern pattern was reported previously (1).

The Type 11-specific probe detected only the 3.2-kb RNA band (Fig. 3, lane 2 ) . Since the 3.7-kb form was not detected with this exon CD-specific probe, even after a longer exposure (not shown), we identified i t as DTH mRNA Type I. Surprisingly, this mRNA is longer than Type 11, even though it does not contain exons C and D. The only explanation for this apparent discrepancy is that TH mRNA Type I must contain an additional 600-base segment located either in the 5'- or in the 3'-untranslated region.

Considering the results of the previous RACE-PCR experiment, the 3'-UTR was a good candidate for this additional 600-base segment. A radioactive probe was synthesized from the region between the first two polyadenylation signals (probe 3'). When hybridized to the same Northern blot, this probe detected only the longer mRNA (Fig. 3, lane 3 1, identifying this 3.7-kb mRNA as the Type I, (Type I with a long 3'-UTR). The major 3.2-kb mRNA observed with the exon CD-specific probe (lane 2 ) is not detected with the 3' probe; thus, this band corresponds to DTH mRNA Type 11, (Type I1 with a short 3'- UTR). Taken together, the results of the Northern and RACE- PCR analyses suggest that Type I1 mRNA uses the most proximal poly(A) site, whereas Type I uses the distal site.

These observations do not rule out the possibility that an additional polymorphism exists at the 5' end of the DTH transcripts. The Drosophila embryonic cDNA library is unidirectional (421, meaning that the cDNA inserts are uniquely ori- ented relative to the plasmid vector. I t was then possible to PCR amplify specifically the 5' end of TH cDNAs from the population of TH inserts in the library (Fig. 4). The primer pairs consisted of 1) a specific TH antisense oligonucleotide

Primer: II I

cDNA: Lib II Lib I I "

1 2 3 4

Exons: B C D E B E

~

a a FIG. 4. PCR mapping of the 5' end of DTH cDNh. Antisense

primers specific for DTH mRNAType I1 (OTH5, exon D, lunes 1 and 2 ) or Type I (OTH6, exons B-E, lunes 3 and 4 ) and a generic primer (ONB5') that anneals to the vector of the cDNA library (pNB40) were used for PCR amplification. The template was either the unidirectional embryonic cDNA library (-1 ng of total DNA, lunes 1 and 3 ) or an isolated DTH cDNA Type I1 clone (2.5 pg of DNA, lunes 2 and 4). Addition of the generic primer (ONB5') was preceded by 15 cycles of primer extension with the TH-specific primer alone. The length of the major amplification products is indicated at the right. Additional minor and smaller bands in lunes 1 and 3 are due either to other less fre- quently used start sites of transcription or more likely to artifactual halts of the reverse transcriptase during library construction.

409 - 195 -

(bP)

- II 409-

- I 195-

1 2 3 4 5 6 1 2 3 4 5 6

EXON: E - RT PRIMER: a

G - * a * *

FIG. 5. Tissue-specific expression of the DTH &As. %tal RNA was extracted from the indicated tissues and submitted to RT-PCR analysis. The primers used for reverse transcription were either a TH exon E (A, OTHI) or a TH exon G ( B , OTH4) antisense primer. The primer pair used for PCR flanked exons C and D as in Fig. 1. Tissue sources for RNA (both in A and B ) : lune 1, larval CNS; lune 2, larval hypoderm; lune 3, adult brain; lane 4, adult head; lune 5, adult thorax; lune 6, adult abdomen. No difference was detected in amplification products between tissues from male and female flies (not shown). The

the PCR products at the left. mRNA species is indicated at the right of each panel and the length of

(either OTH5 or OTH6, see "Experimental Procedures") and 2) a generic primer annealing to the pNB40 vector just upstream from the 5' end of the cDNAinserts (ONB5'). With a TH primer specific for mRNAType I1 (OTH5, annealing to exon D), several fragments were amplified (Fig. 4, lane 1 ), but the longest one is by far the most abundant, showing that nearly all the DTH Type I1 cDNAs in this library have the same 5' end. Fig. 4, lane 2 , shows that a band of exactly the same length is obtained in a control amplification performed with a pure DTH cDNA Type

26564 A Novel and Major Isoform of Drosophila erosine Hydroxylase

TYPE II

BASIC ACIDIC

0 1W 200 300 400 500 AA

FIG. 6. A, structure of the Drosophila TH mRNks. The DTH tran- scripta are drawn to scale (bar = 500 bp). Exon boundaries are represented, and filled boxes indicate the protein coding portion. The exons C and D (speckled bores) are alternatively spliced as a pair, resulting in two types of coding region: with (Type 11) or without (Type I) exons C and D. Exon G has two possible lengths according to the poly(A) signal (asterisks) used, which is either the first ( S , short 3’-UTR) or the third (L, long 3’-UTR). Four types of mRNAs are predicted: Types I,, 11,, I,, and 11,. Two of these are shown shaded, because one (Type I,) has never been detected and the other (Type 11,) is a minor form. Tissue localization of each transcript is indicated. In addition, the mRNA sequences hybridizing to each of the probes used for Northern analysis (I +II, 11, and 3‘, Fig. 3) are represented (horizontal bars). B , structure and acid/ base profile of the Drosophila TH protein isoforms. Regions showing conservation between vertebrates and DTH proteins (1) are shown below the Type I profile. The regulatory and catalytic domains indicated are deduced from corresponding sequences in the vertebrate TH protein (49-51). Serine 32, which is a putative site for CAMP-dependent protein kinase, is found in one of the three conserved regions showing signifi- cant homology with mammalian TH. The speckled bores in TH Type I1 are the alternatively spliced exons C and D. Positions of acidic and basic amino acids in DTH Type I1 are deduced from the cDNA sequence (see Fig. 2). The plot comes from DNA Strider (Christian Marck). The height of each vertical bar represents a different amino acid: for acidic residues, long is Glu and short is Asp; for basic residues, long is Arg, short is Lys, and very short is His. The scale indicates the number of amino acids.

I1 clone. When a TH primer specific for mRNA Type I was used (OTH6, annealing to the exon BE junction), the major band amplified is 154 bp smaller (Fig. 4, lane 3) , as expected if the two types of mRNAs have the same 5’-UTR. One possible prob- lem is that the 3’ end of the primer OTH6 could anneal at low stringency to exon B, which is common to both types of mRNA. However, the control with the pure DTH cDNA Type I1 clone only led to a barely detectable amplification product (lane 4 ) , confirming that the primer OTH6 is rather specific for DTH cDNA Type I in these conditions.

These observations suggest that the 5‘ end of the cDNA clones isolated is the major transcription start site of the DTH gene and that the same site is utilized for transcription of both mRNAType I and 11. We looked for this start site by sequencing the 5’ portion of the DTH gene. As indicated in Fig. 2, a putative TATA box, characteristically surrounded by GC-rich sequences, is appropriately located 25 bp upstream from the end of the cDNAs.

It should be noted that the lengths of the DTH mRNAs without poly(A) tail deduced from the sequence and mRNA end mapping data, 3.11 kb for Type 11, and 3.58 kb for Type I,, agree with the values of 3.2 and 3.7 kb, respectively, estimated by Northern blot (Fig. 3).

l)rosine Hydroxylase mRNAs Type IL and l)pe 11, Are Specifically Expressed in the CNS and Hypoderm,

Respectively Many genes that are alternatively spliced in different tis-

sues, particularly in neural and non-neural tissues, have been identified (reviewed in Refs. 47 and 48). Since TH is expressed both in the CNS and in the hypoderm in Drosophila (see In- troduction), it is reasonable to expect that expression of the two TH mRNA subtypes is tissue-specific. Tissue distribution of the DTH mRNAs was studied by RT-PCR (Fig. 51, using a method that allows determination of which 3‘ end is present in each alternatively spliced mRNA. 3’ end determination was performed by using two different oligonucleotides to prime reverse transcription: OTH1, complementary to exon E, and OTH4, complementary to the distal part of the 3”untranslated region in exon G (see Fig. 2) . OTHl primes all TH mRNA types, whereas OTH4 is specific for TH mRNAs that contain the long 3’-UTR. The RNA extracts from the different tissues were di- vided in two equal portions, and each lot was primed either with OTHl (Fig. 5 A ) or OTH4 (Fig. 5B). The PCR step was performed exactly as described in the legend to Fig. 1, using a primer pair flanking exons C and D.

Priming with a Common TH Primer (Fig. 5A)”AS shown in Figs. 1 and 3, DTH mRNA Type I1 is the major form in whole larvae or adults. Fig. 5.4, lane 2, shows that this is also the only TH mRNA species detected in the hypoderm from third instar larvae. Moreover, in adult tissues, Type I1 is the major form in head, thorax, and abdomen (Fig. 5A), which would be expected from a transcript expressed in the hypoderm. A small amount of mRNA Type I1 is also observed in the CNS from larvae or adult (Fig. 5A, lanes 1 and 3) .

In contrast, DTH mRNA Type I seems to be specifically as- sociated with neural tissue. In adults, it is found mainly in the head and appears to be enriched in dissected adult brain (Fig. 5, A and B , lane 3 ) . In larvae, it is only detected when RT-PCR is performed with RNA extracted from the CNS (lane 1 ) .

Priming with a Long 3’-UTR Primer (Fig. 5B)”Reverse transcription was performed with the distal primer OTH4 to detect specifically mRNA with the longer 3’-UTR (Type L). The major form amplified is DTH mRNA Type I, and amplification products are only observed in samples containing RNAs from neural tissues. Surprisingly, a faint signal corresponding to mRNAType I1 was also obtained in adult head and brain (lanes 3 and 4). This demonstrates the existence of m e 11, (Type I1 with a long 3’-UTR) in neural tissues. This type was not detected by the previous Northern blot (Fig. 21, probably due to its low abundance. No amplification product is detected from hypodermal RNAs (lane 2, 5, and 6). It follows that the form expressed in this tissue is Type 11,. confirming what was deduced from the previous Northern analysis (Fig. 2). The long 3’-UTR is thus specific to the CNS.

These results are summarized in Fig. 6 A . Alternative RNA splicing and polyadenylation choice could generate four forms of TH mRNA Types I,, 11,, I,, and 11,. Types I, and 11, are both found in nervous tissue, I, being more abundant than 11,. The most abundant TH mRNA in the fly is Type II,, which is specifically expressed in the hypoderm. The available evidence suggests that Type Is either is of minor abundance or does not occur in the fly. The length of this form would be 3.4 kb, between that of Type 11, (3.2 kb) and that of Type I, (3.7 kb). Since it is not detected by Northern blot (Fig. 31, it is not an abundant form, although it still could conceivably be present at low levels and partly obscured by Type 11, on the blot. From the PCR experiment of Fig. 5 A , lane 2, it is apparent that no Type I mRNA can be detected in purely hypodermal tissue. Therefore, Type I,, if present, would be restricted to the CNS. However,

A Novel and Major Isoform of Drosophila Qrosine Hydroxylase

Drosophila TH

A B C D E F G

26565

mm 500 bp

H Human TH

1 2 3 4 5 6 7 8 9 1 0 11 1213 14*

coding regions are filled in. Asterisks are polyadenylation signals. SpeckZed boxes correspond to the alternatively spliced exons. In human TH, exon FIG. 7. Comparison of the Drosophila and human TH genes. The genes are drawn to scale (bar = 500 bp). Exons are boxed and the protein

2 as well as a 12-bp segment adjacent to the 3‘ end of exon 1 are alternatively spliced, leading to four mRNA species. In DTH, exons C and D are alternatively spliced as a pair (see Fig. 6A). Homologous points in DTH and HTH are joined by a line when they encode a conserved amino acid and correspond to an exon-intron boundary in one or both of the genes. DTH exon A and HTH exon 1 are not related. The 3‘ end of DTH exon B and the 5’ end of DTH exon E cannot be assigned to corresponding points in the HTH gene because of the lack of homology in these segments (dashed lines). In contrast, both ends of HTH exon 3 are well conserved and are located in DTH exon B and exon E, respectively.

our data show that the more downstream polyadenylation site predominates in the nervous tissue.

Predicted Structural Characteristics of the CNS- and Hypoderm-specific Tyrosine Hydroxylase Enzymes

Two domains can be distinguished in the vertebrate tyrosine hydroxylase protein: a regulatory amino-terminal corresponding roughly to the first 175 amino acids, followed by a catalytic domain (49-51). The homology between the Drosophila and vertebrate enzymes (1) indicates that both domains are conserved in DTH. The proteins encoded by DTH mRNA Type I and Type I1 are predicted to differ, due to the alternative splicing of exons C and D, and this structural difference is restricted to the regulatory domain of the molecule (Fig. 6B).

The regulatory portion of the Type TI DTH contains an additional segment of 71 amino acids located between two regions which are evolutionarily conserved. This additional segment is very acidic and is expected to form a cluster of negative charges (Fig. 6B). The isoelectric point of the regulatory portions is estimated to be 9.0 for TH Type I and 4.7 for Type 11. As discussed below, this observation suggests that the two DTH enzymes have the same hydroxylase activity but possibly differ in their regulatory properties.

DISCUSSION

Diversity and Tissue Distribution of the Drosophila TH mRNAs-Our results show that two isoforms of tyrosine hydroxylase protein are encoded in Drosophila from alternatively spliced exons. Alternative RNA processing of a unique primary transcript generates three types of TH mRNAs that differ in exon composition and 3’-UTR length: types I,, 11,, and 11, (Fig. 6). In addition, evidence is presented for the tissue-specific expression of these transcripts. The single DTH cDNA clone previously reported (1) does not include exons C and D and appears to correspond to mRNA Type I,. This is somewhat surprising because this form of TH mRNA is of low abundance in Drosophila. However, it was isolated by screening an adult head cDNA library, and we have shown that Type I, is present in adult brain (Fig. 5) .

Northern analysis and RT-PCR experiments show that Type 11, is the major form of TH mRNA in Drosophila. It seems to be preferentially expressed in non-neural tissues and may be restricted to the hypoderm. Evidence for the presence of mRNA Type 11, comes from RT-PCR experiments. This form was not detected on Northern blots, probably because of its low abundance. It is expressed as a minor form in the CNS, together with Type I,,.

Several other genes are spliced into distinct mRNAs in neural versus non-neural tissues both in vertebrates and in inver- tebrates. In Drosophila, two forms of dopa decarboxylase

mRNA are generated through tissue-specific alternative splicing of a single primary transcript (8, 9, 32). Consequently, the CNS Ddc enzyme contains an amino-terminal extension of 35 amino acids compared with the hypodermal protein (52). Other known examples in Drosophila include antennapedia (531, Ul- trabithorax (541, and neuroglian (55).

Structure and Alternative Splicing of the TH Primary Dan- script in Drosophila and in Vertebrates-Fig. 7 compares the structure of the Drosophila and human tyrosine hydroxylase genes. The length of the TH gene is similar in Drosophila and in vertebrates; the human TH gene (HTH) encompasses about 8 kb of genomic DNA (35,361 and the rat TH gene 7.5 kb (341, compared with 6.2 kb in Drosophila. The 5’- and 3’-UTRs of the mRNAs are much longer in Drosophila. Thus, the 5’-UTR of bovine TH is only 27 bp (37). Nevertheless, the length of the tyrosine hydroxylase protein is conserved: 498 amino acids for the rat enzyme (331, compared with 508 for DTH Type I, with a sequence identity of about 50% between the two enzymes (1). However, the number of exons is doubled in the vertebrate TH gene: 13 exons in rat and 14 in human (411, compared with 7 in Drosophila (Figs. 2 and 7). The longest evolutionarily conserved portion is in the second half of the protein, the catalytic domain (Fig. 6B). In this region, some of the exon boundaries are precisely conserved in the Drosophila and human TH amino acid sequences (Fig. 8). Thus, the 3‘ end of Drosophila exon E falls at the homologous amino acid as the 3’ end of human exon 9, and the 5’ end of DTH exon G similarly corresponds to the 5’ end of HTH exon 12. DTH exon F is completely conserved, but is contained within two exons in HTH, exons 10 and 11. The sequence of the last two-thirds of DTH exon E is found in HTH exons 5, 6, 7, 8, and 9. In contrast, the amino- terminal portion of the molecule, the regulatory domain, is less well conserved, making it more difficult to assign corresponding human and Drosophila exons. However, two short regions are highly conserved in this domain (Fig. 6B). The first one is found at the very beginning of DTH exon B and HTH exon 3, strongly suggesting that the 5‘ end of exon 3 corresponds to the 5’ end of exon B. The second conserved region is found in DTH exon E and corresponds to the 3‘ end of HTH exon 3 and the 5’ end of HTH exon 4 (Fig. 7).

In vertebrates, alternative splicing of tyrosine hydroxylase only occurs in higher primates (39-41) and is considered to be a relatively recent acquisition in vertebrate evolution. No homology is observed between DTH exons C and D and the two exons alternatively spliced in human TH or any other HTH exons. Moreover, Fig. 7 shows that the alternatively spliced exons are not located in homologous regions in both genes; in HTH, alternative exons are in the first intron, whereas DTH exons C and D are within the equivalent of HTH exon 3. There-

26566 A Novel and Major Isoform of Drosophila Qrosine Hydroxylase

fore, alternative splicing seems evolutionarily unrelated in DTH and HTH. Since the vertebrate version of the gene (i.e. lacking exons C and D) is expressed in Drosophila as mRNA Type I, it is possible that the exons C and D arose during insect evolution. The alternate view, that exons C and D were lost during vertebrate evolution, would predict that these exons might also be found in other lower animals.

The Structural Difference between the Tyrosine Hydroxylase Enzymes Expressed in the CNS and Hypoderm-Strikingly, both in Drosophila and in primates, alternative splicing results in the inclusion or exclusion of short segments in the 5’ regulatory domain of the molecule. The 71-amino acid sequence encoded by the alternatively spliced DTH exons C and D is not related to any other sequence in the data banks. However, it is remarkable that 23 amino acids with acidic residues (aspartate or glutamate) are present in this short segment, with only 1 lysine. This sequence forms a cluster of negative charges in the amino-terminal domain of TH mRNA Type 11, which is clearly apparent in the acid-base diagram of Fig. 6B. Consequently, the regulatory portions of the Type I and Type I1 enzymes are expected to have very different isoelectric points. As mentioned previously, we estimate the PI of the regulatory domain to be 9.0 for Type I and 4.7 for %e 11, in the absence of phosphorylated residues.

No data are available concerning the regulation of tyrosine hydroxylase activity in Drosophila. However, what is known of the regulation of TH in mammals may help to interpret the structural difference between the two DTH isoforms. In vertebrates, tyrosine hydroxylase activity is exquisitely controlled in vivo, subject to down-regulation by the end products of the biosynthetic chain such as dopamine (56, 571, and activated by phosphorylation (reviewed in Ref. 58). Trans-synaptic regulation of the enzyme also occurs at the transcriptional level (59- 61). For example, phosphorylation of Ser4’ by CAMP-dependent protein kinase induces a stimulation of TH activity both in vivo (62) and in vitro (63). The mechanism of this activation is not known, but might be partly explained by the fact that the phosphorylated enzyme is much less sensitive to inhibition by its end product, dopamine (56, 57, 63, 64). In addition, the stimulatory effect of CAMP-dependent phosphorylation on TH activity can be mimicked by adding certain polyanions (such as heparin) to the reaction medium (65). These polyanions bind to the whole enzyme, but not to the active fragment generated after mild proteolysis (66). This fragment is now recognized as the catalytic domain (50, 51, 67), showing that heparin acts by binding to the regulatory domain of the enzyme. Therefore, either CAMP-dependent phosphorylation or polyanion binding at the same time stimulate TH activity and increase the negative charge in the regulatory domain. A more acidic regulatory domain is thus characteristic of the stimulated enzyme.

In DTH, there is a unique consensus site for phosphorylation by CAMP-dependent protein kinase at Ser3’ (RRXS, boxed in Fig. 2). As already indicated by Neckameyer and Quinn (11, this serine is present in one of the conserved regions of the enzyme (Fig. 7) and is likely to be structurally and functionally equivalent to the Ser4’ from rat TH. The cluster of negatively charged amino acids, characteristic of the Drosophila Type I1 enzyme, is inserted just after the well conserved regulatory region that includes SeI.3’ (Fig. 7). We propose that this cluster of negative charges will have a stimulatory effect on tyrosine hydroxylase activity, similar to the effect of heparin on bovine TH activity. The difference is that, in this case, the polyanion is contained within the molecule and induces a permanent reduc- tion in the isoelectric point of the regulatory domain.

Tyrosine hydroxylase catalyzes the rate-limiting step for dopamine synthesis and so must be precisely regulated in the

CNS to respond accurately to the need for neurotransmitter. In contrast, in the insect hypoderm, tyrosine hydroxylase has to produce enormous amounts of dopamine in a short period of time when a new cuticle is synthesized. Inhibition by the end products would clearly be a disadvantage for the fly at these stages, which may explain why the hypodermal TH contains this additional acidic segment.

A Different Polyadenylation Site Is Used for Drosophila TH mRNAs in the Hypoderm and CNS-The results presented suggest that the choice of the polyadenylation site in the TH gene is highly tissue-specific. In the hypoderm, the proximal poly(A) signal is used, resulting in expression of DTH mRNA Type 11,. Conversely, DTH mRNA Type I, and Type 11,, which both contain the long 3’-UTR, are only detected in the CNS.

Although alternative polyadenylation is a common occur- rence, few examples are found in the literature showing tissue specificity in the choice between poly(A) sites located in a single terminal exon. There is one example of alternative splicing and polyadenylation that is strikingly similar to the DTH RNA processing. The Drosophila gene Ultrabithorax expresses two types of mRNA: 1) a 3.2-kb mRNA that contains two alternatively spliced internal mini-exons, has a short 3’-UTR, and is expressed in non-nervous tissues, and 2) a 4.3-kb mRNA that skips one or both of the mini-exons, has a long 3’-UTR and is expressed in the CNS (54, 68).

I t is generally accepted that the cleavage of a transcript at a poly(A) site occurs before completion of transcription (69), from which it can be inferred that the first polyadenylation site has normally an advantage over the more distal ones. Since in the Drosophila CNS the first two poly(A) signals of the TH gene are skipped, most likely a negative control prevents formation of the polyadenylation complex at those sites in the nervous system. This may indicate that the long CNS-specific 3”untranslated segment present in DTH Type I mRNA has an important, but for now totally unknown, function.

Our results reveal an unexpected diversity in Drosophila tyrosine hydroxylase mRNAs and gene products. Further stud- ies that are feasible in this genetically tractable animal will be required to confirm our hypothesis regarding the effects of the alternative exons on DTH enzyme activity and the biological relevance of these observations.

Acknowledgments-We thank D. M. Chikaraishi for the gift of the rat TH cDNA, N. H. Brown for the Drosophila embryonic cDNAlibrary, and N. J. Lewis, M. J. Lundell, and M. Meller for critical reading of the manuscript.

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A Novel and Major Isoform of Drosophila prosine Hydroxylase 26567

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