Ornithine decarboxylase antizyme
Both forms of guf RNA are readily detected although the yield of mature RNA is greater than the precursor transcript in both wild-type and Sxlf4 ovaries. The overall yield from wild-type ovaries was lower for both products, but since the immunoprecipitations and RT-PCRs are not quantitative, this comparison is only speculative. To determine whether Sxl also binds guf RNA in somatic tissues, the immunoprecipitation and RT-PCR performed using embryonic extracts. Sxl also binds to spliced and unspliced guf RNA in embryos (ratio of spliced to unspliced is approximately equal). These data suggest that Sxl may regulate both the splicing and translation of guf (Vied, 2003).
Since Sxl binds to guf RNA in both the soma and germline, RT-PCR amplifications were performed for the four guf transcripts to determine whether any sex-specific products could be detected. guf RNA was analyzed from males and females (whole animals and carcasses) as well as testes and ovaries. For all cDNA types, no significant difference between the sexes was observed. From these data, it is concluded that Sxl does not regulate the alternative splicing of guf RNA in a global manner. Given the general requirement for guf, splicing regulation of guf RNA by Sxl may be restricted to only a subset of cells, such as the early germ cells, making detection of altered transcripts unlikely. Since there are no apparent alternative exons within guf, it is also possible that Sxl causes a retention of the introns leading to the degradation of the RNA. Alternatively, the regulation by Sxl may occur primarily at the level of translation control (Vied, 2003).
While the RNA binding data suggest that guf is a target of Sxl, a genetic interaction between the two genes would strengthen the idea that the two genes have a related function. Therefore the dose of guf was reduced in homozygous Sxlf4 females, which normally have small ovaries of tumorous egg chambers. This was done by using a P-element insertion allele (guf118-3) or a small deletion allele produced by the imprecise excision of the guf118-3 P-element (guflex47; Salzberg, 1996). Reducing guf dose in a homozygous Sxlf4 background rescues the Sxlf4 phenotype. The females lay eggs that hatch and produce adults (Vied, 2003).
Since the original description of guf, an additional gene, SmD3, was found in the intron of guf (Ivanov, 1998). SmD3 encodes a snRNP (small nuclear ribonucleoprotein) common to splicing snRNPs and is transcribed in the opposite direction to guf. More relevant to the existing guf alleles is that the P-element insertions that uncover guf are likely to be affecting both genes. The P-elements are inserted in the 5' UTR of SmD3, and guf deletion alleles derived from their imprecise excision affect the coding sequences of both genes. Additionally, it was recently suggested that the reported phenotype of guf (Salzberg, 1996), as caused by transposon insertions, is primarily due to the alteration of SmD3 (Schenkel, 2002). Since Sxl is a splicing regulator, it was unclear whether the effects on Sxlfs females were a result of reducing guf or SmD3 or both. Additional experments indicate that the rescue is a result of an interaction of Sxl with guf and not with SmD3. Data also indicate that, in the germline, the guf118-3 allele does affect guf expression. Since reducing the dose of guf allows differentiation of Sxl mutant germ cells, the data suggest that Sxl normally functions as a negative regulator of guf. Presumably, the mutant Sxl proteins are unable to properly regulate the splicing and/or translation of guf RNA (Vied, 2003).
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