Invertebrate synapsins: a single gene codes for several isoforms in Drosophila

Abstract

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

Fig. 6.

Schematic of cDNA fragments used as hybridization probes (P1–P3) or expressed as GST fusion proteins inE. coli. Fusion proteins 5′-FP and 3′-FP were used for immunization and production of antisera and monoclonal antibodies in mice. ∼∼∼∼ denotes homology region; I marks the internal stop codon at nt 1965 of cDNA Syn-1; cDNA Syn-1⁺ corresponds to nt 2246–3377;arrows delimit reading frames (stop to stop). B,BamHI; E, EcoRI restriction sites.

Fig. 1.

Nucleotide sequence of Syn-1 (a) and Syn-2 (b) cDNAs and inferred protein sequence (one-letter amino acid code). Bold amino acids (nt 618–1544): homology to C domain of vertebrate synapsins. All critical regions have been verified by genomic sequencing, including the 5′ and 3′ ends (both presumably incomplete) and the two large open reading frames ORF1 and ORF2 delimited by three stop codons (doubly underlined). The two possible translation start codons are underlined (compare Discussion). Intron positions are indicated by arrowheads.Asterisks mark serine and proline repeats. Arrowsare shown below the first amino acids of the 5′- and 3′-fusion proteins (FP), respectively. The following deviations from the cDNA sequence (changes in inferred protein in parentheses) have been noted in the genome: 1451: A to G (N to D); 1875–1877 missing (deletes P); 3284: A to T; 3606–3608: missing (no changes). Differences to Syn-1 are underlined in b. The nucleotide sequence data reported here will appear in the EMBL, Genbank, and DDBJ nucleotide sequence databases under the accession numbers X95453(Syn-1) and X95454 (Syn-2).

Fig. 2.

Sequence comparison of 309 amino acids of the inferred Drosophila synapsin homolog protein (SYN) (1) with the C domain of rat (2) and human (3) synapsin-Ia. Within this domain, 50% of the amino acids are identical (∣), and another 39% are similar (:), allowing for conservative amino acid replacements.Arrowheads indicate intron positions.

Fig. 9.

Top, Domain structure of the four vertebrate synapsins [redrawn according to Südhof et al. (1989)]. Domains A, B, and C are common to all four vertebrate synapsins, domain C is most highly conserved, and domains D–I are more variable.P1–P4 denote phosphorylation sites. Bottom,Drosophila cDNAs Syn-1 and Syn-2 with the possible start codons ATG and CTG as well as the stop codons TAA, TAG, and TGA delimiting the large open reading frames (ORF) (broad lines). Broken lines indicate inferred extensions of cloned sequences. Middle, Present hypothesis, how the mRNAs corresponding to Syn-1 and Syn-2 might be translated into SYN proteins by read-through of the central UAG stop codon (SYN1-RT), respecting this stop (SYN1-S), or using the splice variant (SYN2). For each of these three proteins, the calculated molecular weight is given using the first ATG or the first CTG (numbers in parentheses). Note that only domain C (and its truncated version C* in SYN2) is conserved between vertebrates andDrosophila (indicated by oblique hatching).

Fig. 3.

a, Restriction map and clones of genomic walk including the Syn gene. Polymorphic restriction sites are in parentheses. b, Exon–intron structure of transcript corresponding to Syn-1. Boxesindicate exons. Syn-2 transcript differs from Syn-1 by four bases inserted at the end of exon 10 because of the use of an alternate 5′ splice site of intron 10. Homology region is hatched.White bar marks position of internal TAG stop codon. The two possible translation initiation codons (CTG, nt 174; ATG, nt 356) are indicated by curved arrows (compare Discussion).

Fig. 4.

Sequences of exon–intron boundaries compared with consensus sequence (Senapathy et al., 1990; Mount et al., 1992).Bold letters, Introns; italics: exons. Bases in agreement with consensus are underlined. Intron size and relative agreement are given in parentheses. Alternately spliced base pairs are marked by ∧.

Fig. 5.

In situ hybridization using cDNA fragments P1 + P2 of Figure 6 as digoxygenin-labeled probe and anti-digoxygenin immunohistochemistry (a) or using cDNA Syn-1⁺ as ³⁵S-labeled probe and contact autoradiography (b, c). Expression of theSyn gene is evident for most or all parts of the late embryonic nervous system (a). b, Unstained frozen head section to which probe was hybridized. c, Contact autoradiograph of section in b showing specific signals for most of the brain cellular rind (chitin binds probe unspecifically). Am, Antennal maxillary complex;La, lamina; Lo, lobula; Lp, lobula plate; Me, medulla; PNS, peripheral nervous system; R, retina; Vg, ventral ganglia. Scale bars: 20 μm (a) and 100 μm (b, c).

Fig. 7.

Western blot analysis of Syn gene expression in Drosophila heads and in two transformedE. coli strains, BL21 (B) and DH5α (D). UAG stop codons are read through at low efficiency in DH5α cells because of the presence of a UAG suppressor tRNA in this strain (sup⁺). BL21 is sup⁻. a, mAb SYNORF1 recognizes in sup⁻ E. coli the massively induced fusion protein of 78 kDa (plus degradation products) (induced, B⁺; noninduced, B⁻); in sup⁺ E. coli the 78 kDa protein; and, in addition, the read-through form of ∼150 kDa (induced, D⁺). In fly heads (H), a protein triplet of 70, 74, and 80 kDa and a doublet at ∼143 kDa are recognized. b, Antiserum SYNORF2 recognizes the 66 kDa 3′-fusion protein used for immunization (3′-FP), the read-through form in sup⁺ strains (D⁺), and only the ∼143 kDa protein doublet in head homogenates (H). The massively induced 78 kDa protein does not bind this antiserum but is faintly recognizable in the B⁺ and D⁺ lanes because of weak unspecific staining.c, Semiquantitative analysis of Western blot signals obtained with mAb SYNORF1 from 1/4, 1/2, 1, 2, and 4 heads per lane predict a read-through efficiency of 20–25% in heads.

Fig. 8.

a, Immunohistochemical staining by mAb SYNORF1 of synaptic boutons on a larval body wall muscle preparation. b, Immunohistochemical staining of a horizontal section through an adult Drosophila head using mAb SYNORF1 (dilution 1:4). Most synaptic neuropil is stained strongly.c, Optic lobes stained by using mAb SYNORF1 at 1:150 dilution to show the weaker antibody binding to the lamina (La) and a layer of the medulla (Me, arrowhead). Axons (between neuropil masses) and perikarya of the cellular rind (CR) are almost devoid of staining. d, e, mAb SYNORF1 staining of thoraco-abdominal neuropil (d) and synaptic boutons on a direct-flight muscle (e). Five antisera generated against the 5′-fusion protein (5′-FP) and six antisera against the 3′-FP show staining patterns indistinguishable from those of mAb SYNORF1. AG, Abdominal ganglion;AL, antennal lobe; CC, cervical connective;CR, perikarya of cellular rind; Lo, lobula;LP, lobula plate; LPr, lateral protocerebrum;MsTG, mesothoracic ganglion; MtTG, metathoracic ganglion; PTG, prothoracic ganglion; R, retina. Scale bars: 20 μm (a, c, e) and 100 μm (b, d).