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

Fakultaet fuer Chemie AG,. Molekulare Zellbiochemie, Ruhr-Universitaet, Bochum, Germany.
Gene (Impact Factor: 2.14). 04/2000; 245(1):127-37. DOI: 10.1016/S0378-1119(00)00027-5
Source: PubMed


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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    • "Among them, the following hnRNP proteins are similar to the human hnRNP A/B type, which has two RNA-binding domains and one glycine-rich and M9-like (nuclear shuttling signal) domain: Hrb87F/hrp36 (19); Hrb98DE/hrp38 (20); Squid/hrp40 (21) and Hrb27C/hrp48 (22). Hrb57A/Bancal encodes a homolog of the vertebrate hnRNP K protein (23,24), and Hrp59 encodes a homolog of the vertebrate protein hnRNP M containing three RNA-binding domains (25,26). Hrp36 (27), hrp38 (20), hrp40 (28), hrp48 (22,29) and hrp59 (25) have all been shown to be involved in pre-mRNA splicing, and hrp40 is required for proper RNA localization (21,30,31). "
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    ABSTRACT: The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsr omega-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways.
    Nucleic Acids Research 05/2009; 37(11):3501-13. DOI:10.1093/nar/gkp218 · 9.11 Impact Factor
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    • "Analyses of the proteins associated with the omega speckles have suggested possible function of the hsrω-n transcripts. The following proteins are known to be associated with the nucleus-limited hsrω-n transcripts: (i) various hnRNPs (12,13): Hrp40 (hnRNP A) (41), Hrb87F (hnRNP A1/A2) (42), Hrb57A (hnRNP K) (43), S5 (hnRNP M, H. Saumweber, personal communication), Hrb98DE (44), etc., (ii) other nuclear RNA-binding proteins like NonA, PEP (45), Sxl (46), nuclear non-histone proteins recognized by Q14, Q16, T29, P75 antibodies (12,47), (iii) Snf (45), (iv) Hsp83 (48) and (v) Tpr (45), etc. Most of these proteins (in groups i–iii) are normally associated, in addition to their presence in the nucleoplasmic omega speckles, with many sites on chromosomes, specially those that are transcriptionally active. "
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    ABSTRACT: Exposure of cells to stressful conditions elicits a highly conserved defense mechanism termed the heat shock response, resulting in the production of specialized proteins which protect the cells against the deleterious effects of stress. The heat shock response involves not only a widespread inhibition of the ongoing transcription and activation of heat shock genes, but also important changes in post-transcriptional processing. In particular, a blockade in splicing and other post-transcriptional processing has been described following stress in different organisms, together with an altered spatial distribution of the proteins involved in these activities. However, the specific mechanisms that regulate these activities under conditions of stress are little understood. Non-coding RNA molecules are increasingly known to be involved in the regulation of various activities in the cell, ranging from chromatin structure to splicing and RNA degradation. In this review, we consider two non-coding RNAs, the hsr(omega) transcripts in Drosophila and the sat III transcripts in human cells, that seem to be involved in the dynamics of RNA-processing factors in normal and/or stressed cells, and thus provide new paradigms for understanding transcriptional and post-transcriptional regulations in normal and stressed cells.
    Nucleic Acids Research 02/2006; 34(19):5508-14. DOI:10.1093/nar/gkl711 · 9.11 Impact Factor
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    • "Rhodamine-tagged anti-dig antibody was used to detect the hybridization as described earlier (Prasanth et al. 2000). After the RISH, the squash preparations were processed for immunostaining with antibodies against various RNA-binding proteins like Sxl (Samuels et al. 1994), HRB87F (hnRNP A1 homologue , Hovemann et al. 1991), Hrb57A (hnRNP K homologue , Hovemann et al. 2000), S5 (hnRNP M homologue, H. Saumweber and K. H. Glaetzer, personal communication; Saumweber et al. 1980), Hrp40 (or Squid, hnRNP A1/2 homologue, Matunis et al. 1992), SRp55 (or B52, a 52-kDa RNA-binding protein of the SR family, Champlin et al. 1991) and Rb97D (Heatwole and Haynes 1996), and antibody binding was detected through FITC-conjugated secondary antibody as described earlier (Prasanth et al. 2000). In another set, squash preparations of testes from wildtype or hsrω 05241 or Df(3R)e Gp4 /Df(3R)GC14 flies were directly processed for immunostaining with the antibodies against RNA-binding proteins. "
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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 · 1.09 Impact Factor
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