Molecular Biology Reports 14: 93-94, 1990. 9 1990 Kluwer Academic Publishers. Printed in Belgium.
THE
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DROSOPHILA Hrb LOCI: A FAMILY OF hnRNA BINDING PROTEINS
SUSAN R. HAYNES 1, GOPA RAYCHAUDHURI 2, DIANA JOHNSON 3, SALLY AMERO 2 and ANN L. BEYER ~ 1Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892 USA; 2Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908 USA; ZDepartment of Biology, George Washington University, Washington, DC 20052 USA. In the nucleus, nascent RNA transcribed by RNA polymerase II, termed hnRNA, is associated with a specific set of hnRNA binding proteins, the hnRNP proteins. These proteins have been extensively characterized in vertebrates, particularly mammalian systems, but comparatively little is known regarding the corresponding proteins in invertebrates. We have been characterizing a Drosophila gene family encoding proteins highly homologous to the the A and B proteins of mammalian hnRNP complexes (Haynes et al. 1987, 1990). The proteins encoded by the Drosophila Hrb98DE and Hrb32AB loci are similar in sequence and structural organization to the A and B proteins. That is, the N-terminal halves of the proteins consist of two copies of the highly conserved RNP consensus domain, or RNA recognition motif, containing the RNP octamer sequence. The Drosophila proteins are 77% identical to each other and -58% identical to the rat A1 hnRNP protein in this region. The C-terminal halves are glycine rich (-40% glycine) with interspersed aromatic amino acids. The exact sequences of the glycine rich regions are poorly conserved in the Drosophila proteins and the A and B proteins, but the overall amino acid composition is very similar. The Hrb98DE gene produces eight different transcripts by use of alternative exons and splice acceptor sites, and these transcripts encode four protein isoforms that differ only at the first 16-20 amino acids. Transcripts from both loci are present throughout development. Transcript levels are high in ovaries and early embryos, indicating that the genes are transcribed maternally. Subsequently, most of the maternal transcripts decay and mRNA levels remain low for the remainder of embryogenesis and part
or all of larval development. Transcript levels increase again during pupation. Transcripts encoding each of the four Hrb98DE isoforms are present at all stages of development, as shown by a combination of reverse transcription and PCR analysis of stage-specific poly A § RNA. We have cloned part of the N-terminal half of
Hrb98DE into a #-gal expression vector and raised polyclonal antisera to the fusion protein. We are using these antisera to biochemically characterize hnRNP complexes in Drosophila. Western blots of nuclear extracts from Drosophila Schneider tissue culture cells were probed with the anti-HRB antibody. The antibody detects 3-4 protein bands in the expected 38-40 kDa size range, which resolve into 8-10 distinct spots located in the basic region of 2D gels (see figure). The antiserum cross reacts with the HeLa A and B proteins and with bovine UP1.
Fig. 1. Western blot of Drosophila nuclear extract with anti-HRB antibodies. Nuclear extracts prepared from Drosophila tissue culture cells were fractionated on sucrose gradients. Western blot analysis of gradient fractions showed HRB proteins migrating as a complex with a peak sedimentation coefficient of approximately
94 40S. Prior RNAse treatment of the extract destroys the complex, and HRB proteins then migrate at the top of the gradient. When gradient fractions were probed for the presence of specific polymerase II transcripts, these transcripts were found to migrate with the 40S hnRNP complex. Indirect immunofluorescence analysis of polytene chromosomes shows anti-HRB antibody reacting with puffs and interbands in a pattern very similar to that observed with anti-RNA polymerase II antibody, suggesting that HRB proteins are coincident with sites of active transcription. REFERENCES Haynes, S.R., Rebbert, M.L., Mozer, B.A., Forquignon, F. and Dawid, I.B. 1987. pen repeat sequences are GGN clusters and encode a glycinerich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein. Proc. Natl. Acad. Sci. USA 84:1819-1823. Haynes, S.R., Raychaudhuri, G. and Beyer, A.L. 1990. The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes. Mol. Cell. Biol. 10:316-323.
THE
93
DROSOPHILA Hrb LOCI: A FAMILY OF hnRNA BINDING PROTEINS
SUSAN R. HAYNES 1, GOPA RAYCHAUDHURI 2, DIANA JOHNSON 3, SALLY AMERO 2 and ANN L. BEYER ~ 1Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892 USA; 2Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908 USA; ZDepartment of Biology, George Washington University, Washington, DC 20052 USA. In the nucleus, nascent RNA transcribed by RNA polymerase II, termed hnRNA, is associated with a specific set of hnRNA binding proteins, the hnRNP proteins. These proteins have been extensively characterized in vertebrates, particularly mammalian systems, but comparatively little is known regarding the corresponding proteins in invertebrates. We have been characterizing a Drosophila gene family encoding proteins highly homologous to the the A and B proteins of mammalian hnRNP complexes (Haynes et al. 1987, 1990). The proteins encoded by the Drosophila Hrb98DE and Hrb32AB loci are similar in sequence and structural organization to the A and B proteins. That is, the N-terminal halves of the proteins consist of two copies of the highly conserved RNP consensus domain, or RNA recognition motif, containing the RNP octamer sequence. The Drosophila proteins are 77% identical to each other and -58% identical to the rat A1 hnRNP protein in this region. The C-terminal halves are glycine rich (-40% glycine) with interspersed aromatic amino acids. The exact sequences of the glycine rich regions are poorly conserved in the Drosophila proteins and the A and B proteins, but the overall amino acid composition is very similar. The Hrb98DE gene produces eight different transcripts by use of alternative exons and splice acceptor sites, and these transcripts encode four protein isoforms that differ only at the first 16-20 amino acids. Transcripts from both loci are present throughout development. Transcript levels are high in ovaries and early embryos, indicating that the genes are transcribed maternally. Subsequently, most of the maternal transcripts decay and mRNA levels remain low for the remainder of embryogenesis and part
or all of larval development. Transcript levels increase again during pupation. Transcripts encoding each of the four Hrb98DE isoforms are present at all stages of development, as shown by a combination of reverse transcription and PCR analysis of stage-specific poly A § RNA. We have cloned part of the N-terminal half of
Hrb98DE into a #-gal expression vector and raised polyclonal antisera to the fusion protein. We are using these antisera to biochemically characterize hnRNP complexes in Drosophila. Western blots of nuclear extracts from Drosophila Schneider tissue culture cells were probed with the anti-HRB antibody. The antibody detects 3-4 protein bands in the expected 38-40 kDa size range, which resolve into 8-10 distinct spots located in the basic region of 2D gels (see figure). The antiserum cross reacts with the HeLa A and B proteins and with bovine UP1.
Fig. 1. Western blot of Drosophila nuclear extract with anti-HRB antibodies. Nuclear extracts prepared from Drosophila tissue culture cells were fractionated on sucrose gradients. Western blot analysis of gradient fractions showed HRB proteins migrating as a complex with a peak sedimentation coefficient of approximately
94 40S. Prior RNAse treatment of the extract destroys the complex, and HRB proteins then migrate at the top of the gradient. When gradient fractions were probed for the presence of specific polymerase II transcripts, these transcripts were found to migrate with the 40S hnRNP complex. Indirect immunofluorescence analysis of polytene chromosomes shows anti-HRB antibody reacting with puffs and interbands in a pattern very similar to that observed with anti-RNA polymerase II antibody, suggesting that HRB proteins are coincident with sites of active transcription. REFERENCES Haynes, S.R., Rebbert, M.L., Mozer, B.A., Forquignon, F. and Dawid, I.B. 1987. pen repeat sequences are GGN clusters and encode a glycinerich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein. Proc. Natl. Acad. Sci. USA 84:1819-1823. Haynes, S.R., Raychaudhuri, G. and Beyer, A.L. 1990. The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes. Mol. Cell. Biol. 10:316-323.
