The major nuclear ribonucleoproteins (RNPs) involved in pre-mRNA processing are classified in broad terms either as small nuclear RNPs (snRNPs), which are major participants in the splicing reaction, or heterogeneous nuclear RNPs (hnRNPs), which traditionally have been thought to function in general pre-mRNA packaging. We obtained antibodies that recognize these two classes of RNP in Drosophila melanogaster. Using a sequential immunostaining technique to compare directly the distribution of these RNPs on Drosophila polytene chromosomes, we found that the two patterns were very similar qualitatively but not quantitatively, arguing for the independent deposition of the two RNP types and supporting a role for hnRNP proteins, but not snRNPs, in general transcript packaging.Both heterogeneous nuclear ribonucleoproteins (hnRNPs; reviewed in refs. 1 and 2) and small nuclear ribonucleoproteins (snRNPs; reviewed in ref.3) are deposited cotranscriptionally on eukaryotic RNA polymerase II transcripts (4-8). Whereas the major basic hnRNP proteins have been considered traditionally to function in general pre-mRNA packaging (2, 9), they have been proposed recently to be specific splicing cofactors or to be preferentially associated with splice junction sequences (10-15). snRNPs are major participants in the splicing reaction (3) but have been implicated recently in general packaging as part of a previously assembled unitary processing complex also containing hnRNPs (5, 6). The various proposals predict different amounts and ratios of the two protein types on nuclear pre-mRNA molecules at chromosomal sites of transcription, which is the issue we have addressed by sequential immunostaining.The core hnRNP proteins (A, B, and C proteins of 32-45 kDa) were originally identified as the major proteins that are associated with newly synthesized pre-mRNA (in the form of 30-50S RNP particles) when it is extracted from nuclei (reviewed in refs. 1 and 2). This observation, together with their nuclear abundance, their ability to bind single-stranded nucleic acids regardless of sequence, and their helixdestabilizing properties, led to the notion that these core hnRNP proteins are involved in general pre-mRNA packaging, much as the histones are involved in the general packaging of DNA (1, 2). However, more recent investigations of hnRNP proteins, using in vitro splicing or in vitro RNA binding studies, have suggested that these proteins play a role in the splicing reaction (10-12), that they bind with high affinity to sequences at 3' splice sites (13,14), and that they are dependent on snRNPs for acquisition of a crosslinkable association with RNA (13). These in vitro studies have led to a reappraisal of the independent structural role of hnRNP proteins in pre-mRNA packaging towards a view that they are a few of the many required cofactors for splicing. The simplest version of this view would predict a constant stoichiometry of snRNPs and the core hnRNP proteins on pre-mRNA, in amounts that correlate with the number of splicing sign...
Using an immunochemical approach, we have identified a unique antigen, PEP (protein on ecdysone puffs), which is associated in third-instar larvae with the active ecdysone-regulated loci on polytene chromosomes; PEP is not associated with most intermolt puffs and is found on some, but not all, heat shock-induced puffs. The distribution pattern changes with changing puffing patterns in the developmental program. We have screened an expression library and recovered a cDNA clone encoding PEP. PEP possesses multiple potential nucleic acid-and protein-binding regions: a glycine-and asparagine-rich amino terminus, four zinc finger motifs, two very acidic segments, two short basic stretches, and an alanine-and proline-rich carboxyl terminus. The Pep gene maps by in situ hybridization to the cytological locus 74F, adjacent to the early ecdysone-responsive region; however, the gene is not regulated by ecdysone at the level of transcription. The pattern of Pep expression through development suggests that maternal Pep gene transcripts are supplied to the embryo, and that the abundance of Pep gene transcripts decreases to a lower, fairly constant level thereafter. This unusual protein may play a role in the process of gene activation, or possibly in RNA processing, for a defined set of developmentally regulated loci.
A new family of cyclophilins with an RNA recognition motif (RRM) has members in vertebrates, roundworms and flatworms. We have identified a Drosophilacyclophilin, Dcyp33, with a high degree of amino acid sequence identity and similarity with other members of the family. Dcyp33 interacts through its RRM domain with the third PHD finger of trithorax. This interaction is conserved in the human homologues of these proteins, Cyp33 and MLL. Over expression of Dcyp33 in DrosophilaSL1 cells results in down-regulation of AbdominalB Hoxgene expression, mirroring the effect of human Cyp33 on the expression of human HOXgenes.
The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.
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