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Analysis of the Cis-Acting Requirements for Germ-Line-Specific Spllcing of the P-Element ORF2-ORF3 Intron

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Analysis of the Cis-Acting Requirements for Germ-Line-Specific Spllcing of the P-Element ORF2-ORF3 Intron Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of the cis-acting requirements for germ-line-specific spllcing of the P-element ORF2-ORF3 intron Frank A. LaskiL3 and Gerald M. RubinL2 1Department of Biochemistry and 2Howard Hughes Medical Institute, University of California, Berkeley, California 94720 USA P-element transposition is limited to the germ line because the element's third intron is only spliced in germ line cells. We show that a 240-bp fragment containing this 190-bp intron can confer germ line specificity when placed in the context of another gene. We find that the c/s-acting regulatory sequences required for germ line regulation map near to, but not at, the 5' or 3' splice junctions. [Key Words: Transposable element~ P element~ RNA splicing~ intron] Received January 24, 1989; revised version accepted March 4, 1989. P elements are a family of transposable elements found ORF2 to ORF3 is the sole basis for the germ line restric- in Drosophila melanogaster. They have been shown to tion of P-element transposition {see Fig. 1 ). be the causal agents of P-M hybrid dysgenesis, a syn- Although knowledge about the biochemistry of drome whose traits include high rates of sterility, muta- splicing is progressing rapidly {for review, see Sharp tion, and chromosomal rearrangements {Kidwell et al. 19871 Green 19861 Padgett et al. 1986} and a number of 1977~ for review, see Engels 19831. Autonomous P ele- altemative splices have been identified {for review, see ments encode transposase, a trans-acting function that Breitbart et al. 1987}, little is known about the mecha- is necessary for P-element transposition and excision nism{s} involved in the regulation of an altemative {Spradling and Rubin 1982~ Engels 1984}. P elements splice. Why are some introns spliced in all tissues at all have been analyzed at the molecular level and found to times while others show specificity? What cis-acting se- range in size from <500 bp to the 2.9-kb autonomous P quences in the pre-mRNA are required for this speci- element {O'Hare and Rubin 1983}. A typical P strain ficity.~ carries 30-50 P elements, of which approximately one- A parasitic element is under strong selective pressure third are 2.9 kb. The short nonautonomous P elements to evolve a mechanism to limit its transposition to the do not encode transposase activity but can be mobilized germ line so that its spread through a population would in trans when a source of transposase is supplied. P-ele- not be impeded by needlessly lowering the viability of ment-mediated germ line transformation {Spradling and its hosts. Although we know that P elements have ac- Rubin 19821 Rubin and Spradling 1982) allows one to complished this objective by evolving a germ-line-spe- introduce a mutated P element back into flies where the cific splice, we do not understand how the tissue speci- mutation's effect on P-element functions can be as- ficity of this splice is achieved. Three classes of models sessed. Using this strategy, Karess and Rubin 11984} have can be envisioned. First, it may be that the ORF2-ORF3 shown that all four open reading frames IORFs} of the P intron is not spliced in somatic cells simply because the element are part of one cistron that encodes transposase. splice recognition sequences are hidden by the tertiary P-element transposition is limited to the germ line. structure adopted by the P-element transcript. In this We have shown that this germ line specificity is model, a germ line factor, which itself may play no role achieved by a germ-line-specific splice that is required to in the splicing of host transcripts, is postulated to unfold join ORF2 with ORF3 {Fig. 11 Laski et al. 19861 Rio et al. the transcript and expose the intron. In the second class 19861. Mutations that alter the consensus 5' or 3' splice of models, a somatic factor specifically inhibits the sites of this intron abolish transposase production. ORF2-ORF3 splice. According to the third model, the Moreover, a mutation that precisely deletes the intron normal splicing machinery is incapable of splicing the produces transposase in somatic tissues as well as the intron and requires additional positive acting factorIsJ germ line. Thus, we were able to conclude that the in- present only in the germ line. ability of somatic cells to splice the intron that joins In this paper we show that the 190-bp ORF2-ORF3 intron retains its germ line specificity when placed, as 3Present address: Department of Biology and Molecular Biology Insti- part of a 240-bp P-element fragment, into the context of tute, University of California, Los Angeles, California 90024 USA. a different gene. We also report the results of our initial 720 GENES & DEVELOPMENT 3:720-728 © 1989 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/89 $1.00 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press P-element germ-line-specific splice pRF0, t ORF1 ~ ORF2 ~ ~ ORF3 ~ third possibility is that a deletion will destroy the in- tron, preventing it from being spliced in any tissue. Al- somatic mRNA UGA though this result provides data on what is required for a A #~ I 66kd ~~~:~~~~~ t AAAA correct splice, it would not supply any direct informa- tion about what sequences are responsible for germ line germline mRNA UAA specificity. 87kd k~ii~i~i~i~i~i~i~:~i~!iiii~i~i~i~`~~`~~`~~`~`~`~`~`~`~`~`~`~ AAAA Transformant lines were assayed first for transposase activity in the germ line using the singed-weak (sn w) Figure 1. Structure of P-element mRNA and proteins. There assay (see Methods). The sn TM allele of the singed bristle are two major P-element RNA transcripts, the 2.5-kb message locus results from the insertion of two nonautonomous made in somatic cells and a germ-line-specific message that P elements in inverted orientation (Roiha et al. 1988). In contains a third splice that connects ORF2 to ORF3 in frame. the absence of P-element transposase the sn TM locus is The 2.5-kb somatic message encodes a 66-kD polypeptide, whereas the germ-line-specific message encodes the 87-kD very stable (all of the progeny are sn~); however, when a transposase protein (Laski et al. 1986; Rio et al. 1986). source of transposase is provided in trans, sn W mutates at a high frequency to two new phenotypes (singed-plus, sn ÷, and singed-extreme, sne; Engels 1984). As seen in mapping of the cis-acting sequences in the pre-mRNA Figure 2, both lines carrying the control construct, wild- that regulate the tissue specificity of splicing. We find type Pc[ry], have high levels of germ line transposase ac- that these sequences appear to map near, but not at, the tivity (46 and 11% of the progeny of flies that carried splice junctions. These results argue against the splice both Pc[ry] and the sn W allele were either sn~ or sn+). being regulated by the tertiary structure of the message. The P[ry(A2-3)} lines also have high levels of germ line activity. The presence of transposase activity in the soma was tested using the P[w] transformant P[w[A)]038 Results (Laski et al. 1986; see Methods}. P[w~A)]038 is homo- There must be cis-acting sequences in the P-element zygous for the white null allele w Ills but has red eyes pre-mRNA that allow the splicing machinery to distin- because it is transformed with a wild-type copy of the guish between the ORF2-ORF3 intron and the other white locus. The wild-type white locus is carried within two P-element introns. Our experiments are designed to a nonautonomous P-element vector, and thus can excise identify these sequences to elucidate their mode of ac- only if transposase is provided in trans {Levis et al. tion. 1985}. Individual somatic excision events generate easily scored white patches in the red eye (Laski et al. 1986). The eyes of flies having one copy of Pc[ry] and P[w] never Most of the ORF2-ORF3 intron sequences are not show such patches (Laski et al. 1986; Fig. 2). However, required for its germ-line-specific splicing flies having one copy of P[w] and P[ry(A2-3)] have high To map whether or not the cis-acting regulatory se- rates of somatic excision. All of the eyes of this genotype quences reside within the third (ORF2-ORF3)intron of examined (a total of 312 eyes from the three transfor- the P element, the deletions diagramed in Figure 2 were mant lines} had a minimum of five patches per eye, al- constructed in Pc[ry]. Pc[ry] contains an active P ele- though most of the eyes had many more excision events. ment marked with rosy (ry), an eye-color gene that en- When the transposase assays were applied to flies car- codes the enzyme xanthine dehydrogenase. It has been rying the deletion mutants, we found that three of the shown that on transformation Pc[ry] acts like a wild- deleted P elements (A1960-2030, A2000-2070, and type P element in Drosophila, producing high levels of A2027-2103) are primarily germ line specific. All three transposase activity in the germ line but none in the were able to transpose stably into Drosophila without soma (Karess and Rubin 1984). Using Pc[ry] we demon- the benefit of a helper P element, and the resulting strated previously that mutations destroying either the transformants had sn w destabilizing activity in the germ 5' or 3' splice junctions of the ORF2-ORF3 intron de- line (Fig.
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