Browsing by Author "T.K. Rajendra"

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Developmental regulation and complex organization of the promoter of the non-coding hsrω gene of Drosophila melanogaster
(Indian Academy of Sciences, 2001) S.C. Lakhotia; T.K. Rajendra; K.V. Prasanth
The nucleus-limited large non-coding hsrω-n RNA product of the 93D or the hsrω 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 hsrω05241 allele due to insertion of a P-LacZ-rosy+ transposon at - 130 bp position of the hsrω promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsrω promoter upstream of the LacZ reporter. The hsrω 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 hsrω05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsrω 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 hsrω 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 hsrω 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.
Male sterility associated with overexpression of the noncoding hsr ω gene in cyst cells of testis of Drosophila melanogaster
(Indian Academy of Sciences, 2001) T.K. Rajendra; K.V. Prasanth; S.C. Lakhotia
Of the several noncoding transcripts produced by the hsrω gene of Drosophila melanogaster, the nucleus-limited > 10-kb hsrω-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsrω-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 hsrω05241 enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsrω05241 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 hsrω-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 Hrp57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsrω-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, SRpSS and Rb97D) were not affected by hsrω 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 overcxpression of hsrω 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 hsrω05241 mutant testes. These results further support a significant role of the noncoding hsrω-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.
Omega speckles - A novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila
(2000) K.V. Prasanth; T.K. Rajendra; A.K. Lal; S.C. Lakhotia
Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsrω-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 hsrω-n transcripts localized was the 93D locus or the hsrω 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 hsrω-n transcripts localizing to the hsrω 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 omega speckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsrω-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsrω transcripts. The omega speckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the omega speckles. The speckled distribution of hnRNPs was completely disrupted in hsrω nullosomics. We conclude that the hsrω-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.
The non-coding transcripts of hsr-omega gene in Drosophila: Do they regulate trafficking and availability of nuclear RNA-processing factors?
(1999) S.C. Lakhotia; Pritha Ray; T.K. Rajendra; K.V. Prasanth
The 93D or hsr-omega (hsrω) is an unusual non-protein-coding gene with multiple transcription products which are dynamically expressed in most cell types of Drosophila melanogaster and this gene, besides being a member of the heat shock gene family, is uniquely induced in polytene cells by a variety of amides. The various aspects of this gene's organization, regulation and inducible properties are briefly reviewed. Recent data in our laboratory show that absence of the hsr-omega transcripts because of nullosomy or over-expression of the these transcripts in specific cell types due to mutation in the promoter region of this gene results in specific phenotypes. It is known from several earlier and our recent studies that in unstressed cell nuclei a variety of hnRNA binding proteins (hnRNPs) associate with many chromosomal sites, including the 93D, and with extra-chromosomal speckles where the hsr-omega transcripts also co-localize. Following heat shock and other stresses, the bulk of these proteins and the hsr-omega nuclear (hsrω-n) transcripts get localized to the 93D site. We propose that one of the important functions of the hsrω-n transcripts is to act as a 'sink' for at least some members of the hnRNPs and related proteins so that any increase or decrease in the abundance of these nuclear transcripts correspondingly modifies the 'sink' size, which in turn affects the availability of such proteins in active nuclear compartments and regulates the nuclear RNA processing activity. It appears that non-availability or over-abundant availability of these transcripts disrupts the regulated and fine-tuned balance of the various RNA-processing factors resulting in trans-dominant mutant phenotypes. We believe that binding with specific proteins and consequent regulation of their activity may be a common feature of the functions of non-protein coding genes.