Functionally antagonistic sequences are required for normal autoregulation of Drosophila tra-2 pre-mRNA splicing

Abstract

Expression of functional TRA-2 protein in the male germline of Drosophila is regulated through a negative feedback mechanism in which a specific TRA-2 isoform represses splicing of the M1 intron in the TRA-2 pre-mRNA. We have previously shown that the mechanism of M1 splicing repression is conserved between distantly related Drosophila species. Using transgenic fly strains, we have examined the effects on regulation of mutations in two conserved features of the M1 intron. Our results show that TRA-2-dependent repression of M1 splicing depends on the presence of a suboptimal non-consensus 3' splice site. Substitution of this 3' splice site with a strong splice site resulted in TRA-2 independent splicing, while substitution with an unrelated weak 3' splice site was compatible with repression, implying that reduced basal splicing efficiency is important for regulation. A second conserved element internal to the intron was found to be essential for efficient M1 splicing in the soma where the intron is not normally retained. We show that the role of this element is to enhance splicing and overcome the reduction in efficiency caused by the intron's suboptimal 3' splice site. Our results indicate that antagonistic elements in the M1 intron act together to establish a context that is permissive for repression of splicing by TRA-2 while allowing efficient splicing in the absence of a repressor.

Figure 1

Replacement of the tra-2 M1 intron with the intron from the ftz gene. (A) A schematic showing the predicted 5′ end of male germline tra-2 pre-mRNA in which M1 intron of tra-2 has been replaced with the constituitively spliced intron of the ftz gene (ftz IVS). Also shown is the relative location of the 7 nt insertion downstream of the 3′ splice site, the transgene probe and the positions of the PCR primers (X3S and X5/X6). Male germline specific transcripts initiate within exon 3 of the tra-2 gene. (B) A Southern blot of low-cycle RT–PCR products from male tra-2/ftzIVS transgenic flies hybridized with a ³²P-end-labeled transgene-specific oligonucleotide containing the 7 nt insertion is shown (lanes 2, 4, 6, 8). Positions of products corresponding to different spliced forms are indicated on the right. Products from PCR with material for mock reverse transcriptase reactions (RT) in which enzyme was omitted are also shown (lanes 1, 3, 5, 7). Samples denoted ‘–’ for TRA-2 are derived from males homozygous for a strong loss of function allele (tra-2^B) that contains a nonsense mutation located within the TRA-2 RNA binding domain. Samples denoted ‘+’ for TRA-2 are derived from sibling heterozygous mutant males (tra-2^B/+). The lanes marked ‘parental’ derive from the strain used for microinjection (w¹¹¹⁸/B^sY; tra-2^B/CyO) which contains no transgene. (C) RT–PCR products from the same RNA samples hybridized with a probe from the M1 intron to confirm that endogenous transcripts undergo tra-2-dependent M1 splicing repression.

Figure 2

Substitutions of sequences near the M1 3′ splice site. (A) Schematic showing the M1 intron (line) and sequences of relevant 3′ splice sites. The 3′ intron sequences present in the ftz3′, CAG3′ and mhce 3′ transgenes are shown. Runs of three or more consecutive pyrimidines are underlined. (B) Southern blots of low-cycle RT–PCR products of RNAs from males of various fly strains. The primers used and the labeling of lanes are as described in Figure 1. Products in lanes 1–4 are derived from RNA of tra-2^B/tra-2^B and tra-2^B/+ of the non-transgenic parental strain and were hybridized with an oligonucleotide probe specific for the endogenous tra-2 gene lacking the 7 nt insertion in exon 4. Products in lanes 5–16 are derived from the various transgenic strains indicated and were hybridized with a transgene-specific oligonucleotide. Expected mobilities of products from spliced and unspliced RNA are indicated by arrows on the right. The faint lower mobility band appearing in lanes 1, 2, 11 and 12 is not dependent on reverse transcriptase and corresponds in size to the products expected from contaminating genomic DNA.

Figure 3

Mutation of a conserved intron sequence decreases M1 splicing in the soma. (A) Schematic showing the sequence and location of the conserved VC element (black box) in the M1 intron (line). The sequence of the VC element and the substitution mutation (VCsub) is shown. (B) Low cycle RT–PCR was performed as in Figure 1B. RT–PCR products were hybridized with oligonucleotides specific for either endogenous or transgene-derived RNA. Expected mobilities and the structure of spliced and unspliced products are shown to the right of the panels. After hybridization with the probe for endogenous transcripts the same blot was stripped and used to detect products from the transgene. Endogenous M1 containing RNA is observed only in wild-type males (upper panel, lanes 7 and 11), but M1-containing RNA derived from the transgenes is observed in both wild-type (lower panel, lanes 7 and 11) and tra-2^B mutant males (lower panel, lanes 5 and 9). (C) Northern blot analysis of poly(A)+ RNA from flies expressing the VCsub mutation. M1 intron containing transcripts were detected using a ³²P-labeled in vitro transcribed antisense M1 RNA. The smaller, TRA-2-dependent RNA corresponds to the normal germline M1 containing RNA and is observed in both the parental (lanes 1 and 2) and transgenic flies (lanes 3 and 4). The larger RNA which is independent of TRA-2 function, is observed at high levels in transgenic flies and at much lower levels in the parental strain. This RNA corresponds in size to transcripts initiating at the somatic transcription initiation site as indicated on the right of the figure. As a loading control the blot was rehybridized with a probe for transcripts from the ribosomal protein 49 (rp49) gene. Note that lane 4 is underloaded.

Figure 4

The VC element is necessary for the efficient removal of the M1 intron in the soma. (A) Schematic diagram of an M1-containing somatic tra-2 RNA is shown. Boxes indicate exons and arrows above indicate both the somatic transcription start site at the beginning of exon 1 and the germline transcription start site within exon 3. PCR primers designed to specifically recognize cDNA generated from the longer somatic tra-2 RNAs are indicated below the RNA. Primer X2S is upstream of the germline-specific transcription start site and primer X5/X6 spans the splice junction between exons 5 and 6. (B) Low cycle RT–PCR analysis of somatic RNA extracted from male fly carcasses from which the testes had been removed by dissection are shown. The flies examined expressed RNA from either the P[VCdel] or the P[VCsub] tra-2 transgenes. Endogenous and transgene RT–PCR products were distinguished with specific oligonucleotide probes as in previous experiments. Note that in all samples the band corresponding to somatic M1-containing RNA comprises a significantly higher fraction of the transgene transcripts compared to endogenous gene transcripts. As observed in previous studies, somatic M1 splicing is independent of the endogenous tra-2 genotype.

Figure 5

Substitution of a strong polypyrimidine tract/3′ splice site into the M1 intron alleviates the somatic requirement for the VC element. M1 splicing in flies expressing P[VCsub,ftz3′] transgene is analyzed by low cycle RT–PCR and Southern blotting. In this transgene the VCsub mutation is combined with a substitution of the strong ftz 3′ splice site. Products from endogenous and transgene transcripts were detected using specific oligonucleotide probes as in other experiments. Expected mobilities of spliced and unspliced products are shown schematically on the right of the panels. Intron-containing transcripts were detected from the endogenous gene (upper panel, lanes 3 and 7) but not from the transgene (lanes 5 and 7) in the presence or absence of functional TRA-2 protein.

Figure 6

Comparison of 3′ splice site regions of introns that undergo splicing repression. The 3′ splice site regions of five introns which are retained in mature mRNA under specific developmental conditions or in response to known regulatory factors are shown in comparison to the D.melanogaster consensus sequences. The splice junctions are indicated by a forward slash and runs of three or more pyrimidines are underlined. Three of the five sequences (cyclin B and two introns from the SWAP gene) lack a polypyrimidine tract of >4 nt near the 3′ splice site. A fourth sequence (msl-2) has a polypyrimidine tract starting 16 nt upstream of the splice site and this spacing has been shown to reduce recognition of the splice site by U2AF in a manner important for splicing repression (46). Splicing of the intron from P element pre-mRNA is known to be repressed by complexes formed near the 5′ splice site of that intron (35).