The protein Hrb57A of Drosophila melanogaster closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins.
ABSTRACT The hnRNP K protein is among the major hnRNA-binding proteins with a strong preference for cytidine-rich sequences. We have cloned a Drosophila hnRNP protein closely related to this vertebrate protein. The protein first identified by the monoclonal antibody Q18 is encoded by a gene located in 57A on polytene chromosomes and has been consequently named Hrb57A. The amino acid sequence of the Hrb57A KH domains and their overall organisation in the protein are remarkably similar to the vertebrate proteins. As the hnRNP K in vertebrates the M(r) 55 000 Drosophila Hrb57A/Q18 protein strongly binds to poly(C) in vitro and is ubiquitously present in nuclei active in transcription. On polytene chromosomes it is found in many puffs and minipuffs. Hrb57A/Q18 specifically coprecipitates four other proteins: Hrb87F/P11 a Drosophila hnRNP A1 homologue, the hnRNA-binding protein S5, the RNA recognition motif-containing protein NonA and the RNA-binding zinc finger-containing protein on ecdysone puffs PEP/X4.
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ABSTRACT: 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.Journal of Genetics 08/2001; 80(2):97-110. DOI:10.1007/BF02728335 · 1.01 Impact Factor
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ABSTRACT: Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsromega-n) transcripts were present in the form of many small speckles. These speckles, which we name 'omega speckles', were distributed in the interchromatin space in close proximity to the chromatin. The only chromosomal site where hsromega-n transcripts localized was the 93D locus or the hsromega gene itself. The number of nucleoplasmic speckles varied in different cell types. Heat shock, which inhibits general chromosomal transcription, caused the individual speckles to coalesce into larger but fewer clusters. In extreme cases, only a single large cluster of hsromega-n transcripts localizing to the hsromega locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuclear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and S5 revealed that, in all cell types, all the hnRNPs gave a diffuse staining of chromatin areas and in addition, were present as large numbers of speckles. Colocalization studies revealed an absolute colocalization of the hnRNPs and the omegaspeckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsromega-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsromega transcripts. The omegaspeckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the &ohgr; speckles. The speckled distribution of hnRNPs was completely disrupted in hsromega nullosomics. We conclude that the hsromega-n transcripts play essential structural and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus.Journal of Cell Science 11/2000; 113 Pt 19:3485-97. · 5.33 Impact Factor