Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

Developmental regulation and complex organization of the promoter of the non-coding hsr(omega) gene of Drosophila melanogaster.

The nucleus-limited large non-coding hsr(omega)-n RNA product of the 93D or the hsr(omega) gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsr(omega) 05241 allele due to insertion of a P-LacZ-rosy+ transposon at -130 bp position of the hsr(omega) promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsr(omega) promoter upstream of the LacZ reporter. The hsr(omega) gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsr(omega) 05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsr(omega) transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsr(omega) RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsr(omega) gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.