Rajendra T K, Prasanth K V, Lakhotia S C
Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India.
J Genet. 2001 Aug;80(2):97-110. doi: 10.1007/BF02728335.
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.
在果蝇的hsromega基因产生的几种非编码转录本中，细胞核内大于10 kb的hsromega - n转录本与不均一核RNA结合蛋白（hnRNPs）共定位，形成精细的核质ω斑点。我们早期的研究表明，非编码的hsromega - n转录本动态调节hnRNPs在活跃（与染色质结合）和非活跃（在ω斑点中）区室中的分布。在这里我们表明，在hsromega05421；增强子陷阱品系中该基因启动子（-130 bp处）的P转座子插入对雄性或雌性的活力或表型没有影响，但插入纯合雄性不育。hsromega05421；纯合果蝇的睾丸含有不活动的精子，而它们的精囊是空的。RNA:RNA原位杂交显示，突变雄性果蝇睾丸中的体细胞性囊细胞含有显著更多的hsromega - n转录本，并且与其他细胞类型中典型的精细ω斑点不同，它们显示出大的ω斑点簇，这通常在热休克后出现。在性囊细胞中表达的两种hnRNPs，即HRB87F和Hrb57A，也与hsromega - n转录本平行在这些细胞中形成大的簇。P转座子插入的完全切除恢复了雄性生育力以及性囊细胞中ω斑点的精细斑点模式。这两种hnRNPs和其他几种RNA结合蛋白（Hrp40、Hrb57A、S5、Sxl、SRp55和Rb97D）在成年睾丸的任何减数分裂阶段的原位分布模式都不受hsromega突变的影响。然而，本研究揭示了在初级精母细胞中意外存在（在野生型以及突变体中）功能性形式的Sxl，并且在第一次减数分裂后期末期沿着收缩纺锤体HRB87F有异常分布。似乎启动子区域的P转座子插入导致性囊细胞中hsromega的失调过表达，这反过来导致hnRNPs的过度隔离以及在这些细胞核中形成大的ω斑点簇。hnRNPs可用性的相应限制可能会反式显性影响性囊细胞中其他前体mRNA的加工。我们认为，由于异常的hnRNP分布导致的性囊细胞活性受损是hsromega05421；突变睾丸中精子个体化失败的原因。这些结果进一步支持了非编码的hsromega - n转录本在基本细胞活动中的重要作用，即调节活跃（与染色质结合）和非活跃（在ω斑点中）区室中hnRNPs的可用性。
