Transgene analysis proves mRNA trans-splicing at the complex mod(mdg4) locus in Drosophila

Abstract

The Drosophila BTB domain containing gene mod(mdg4) produces a large number of protein isoforms combining a common N-terminal region of 402 aa with different C termini. We have deduced the genomic structure of this complex locus and found that at least seven of the mod(mdg4) isoforms are encoded on both of its antiparallel DNA strands, suggesting the generation of mature mRNAs by trans-splicing. In transgenic assays, we demonstrate the ability of Drosophila to produce mod(mdg4) mRNAs by trans-splicing of pre-mRNAs generated from transgenes inserted at distant chromosomal positions. Furthermore, evidence is presented for occurring of trans-splicing of mod(mdg4)-specific exons encoded by the parallel DNA strand. The mod(mdg4) locus represents a new type of complex gene structure in which genetic complexity is resolved by extensive trans-splicing, giving important implications for genome sequencing projects. Demonstration of naturally occurring trans-splicing in the model organism Drosophila opens new experimental approaches toward an analysis of the underlying mechanisms.

Figure 1

Sequence comparison of new Mod(mdg4) protein isoforms. All protein sequences start with amino acid position 403. The most N-terminal 402 aa common to all Mod(mdg4) isoforms are not listed. The conserved C-terminal C₂H₂ consensus sequence is present in all newly identified isoforms. Strongly conserved amino acid positions are shown in bold. Alignment was performed with the clustal x program (16).

Figure 2

Genomic region of the mod(mdg4) complex and the exon/intron structure of identified mRNAs. The alternative 3′-splice site at the exon 4 boundary is used to generate all mature mRNAs indicated by the molecular weight of the deduced proteins. Exons shown below the physical map are encoded by the same DNA strand, whereas those encoded by the antiparallel DNA strand are shown above the physical map. Direction of transcription is indicated by arrows. Untranslated regions are shown as open boxes and translated regions as black boxes. Note the overlap of specific exons five of isoforms mod(mdg4)-55.1 and -53.1 within their 3′-untranslated region and the differential splicing within the specific exons 5 of mod(mdg4)-55.7 and -52.2; -54.6, -56.3, -54.2 and -46.3; -58.6 and -54.1. Restriction sites indicated in the physical map are B, BamHI; X, XhoI; H, HindIII; R, EcoRI; P, PstI; S, SalI; and K, KpnI.

Figure 3

Results of RT-PCR experiments demonstrating the existence of selected mature mRNAs in poly(A)⁺ RNA isolated from early embryos (0–3 h). A primer deduced from common exon 4 (E4-F) was used as forward primer and specific backward primers deduced from the alternative exons 5 indicated above the Upper panel were used for PCR. Specific exons encoded by the same DNA strand as common exons 1–4 are indicated by (+) and those encoded by the antiparallel DNA strand are indicated by (−). PCR-fragments obtained with the same pairs of primers and the corresponding cDNA clones as DNA template are shown in the Lower panel. The expected fragment size for each isoform is indicated at the bottom.

Figure 4

Alignment of 3′-intron sequences upstream of the specific exons 5 of all identified alternatively spliced mod(mdg4) mRNAs. All sequences contain the conserved dinucleotide AG at the 3′-boundary. Pyrimidine-rich tracts are underlined and the putative branch point is indicated by a small bold letter. Specific exons encoded by the same DNA strand as exons 1–4 are indicated by (+) and those encoded by the cDNA strand by (−).

Figure 5

Transgenic assay to demonstrate the existence of trans-splicing at mod(mdg4). Schematic representation of the trans-splicing assay in case of a second chromosomal transgene. Common exons of mod(mdg4) are expressed from the endogenous locus producing mRNA a. The sequence-tagged specific exon mod(mdg4)-55.1 is expressed under control of GAL4 binding to the UAS upstream of the transgene, producing mRNA b. The mature trans-spliced mRNA is detected by RT-PCR with the indicated pair of primers.

Figure 6

RT-PCR demonstrating trans-splicing of endogenous mod(mdg4) common exons 1–4 and tagged-specific mod(mdg4) exons expressed from a transgene. (A) Expression of the tagged exon mod(mdg4)-55.1 is driven by crossing heterozygous transgenic lines to homozygous nanos-GAL4-producing driver lines. The amplicon of the expected size is only obtained in case of trans-splicing of endogenous exon 4 and tagged exon 5 expressed from the transgene. The primer E4-F and tag1-back have been used for PCR. As a control for RT-PCR a backward primer complementary to exon 5 upstream of the sequence tag was used in combination with E4-F (Lower). (B) RT-PCR results obtained with transgenic lines expressing the tagged-specific exon mod(mdg4)-53.1 under control of the nanos GAL4-driver line. The tag2-back primer has been used in combination with primer E4-F. Only in case of correct trans-splicing of the two independent transcripts, the expected amplicon is obtained. No amplicon is obtained in absence of the transgene. For control of RT-PCR the same primer pair as in A was used (Lower). (C) Demonstration of a sequence element with putative promoter activity upstream of specific exon 5 of mod(mdg4)-55.1 contained in the transgene. RT was performed with total RNA from females containing the transgenes TG-1 to 4 (pUAST-55.1-tag1-tag2–53.1) and TG-A and -B (pUAST-53.1-tag2-tag1–55.1) in the absence of a GAL4-driver element. For PCR the forward primer E4-F and the back primer tag1-back were used. In all transgenic lines, a PCR fragment of the expected size was obtained. Quantitative differences among the transgenic lines could be because of position effects. No fragment was amplified with total RNA isolated from the strain w¹¹¹⁸. As a control for RT-PCR, the backward primer deduced from the specific exon mod(mdg4)-55.1 upstream of the sequence tag was used in combination with the E4-F primer.

Figure 7

The model demonstrating different mRNA trans-splicing events at the mod(mdg4) locus. Common exons 1–4 located at the 5′ end of the locus are supposed to be strongly transcribed as a unique transcription unit. Additional transcription units containing one (A) two (B and C) or even more (not shown) isoform-specific exons are initiated at the same coding strand (A) or at the antiparallel DNA strand (B/C). In case of mRNAs containing two or more specific exons, different 5′ splice sites can be used, resulting in the generation of different mRNA variants (B vs. C). The relative amount of single mod(mdg4) protein isoforms could be regulated by the rate of transcription initiation, by the efficiency of trans-splicing, and by the stability of the resulting mRNA.