The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes.

Haynes, Susan R.; Raychaudhuri, G; Beyer, Ann L.

doi:10.1128/mcb.10.1.316

Cited by 53 publications

(65 citation statements)

References 47 publications

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“…In contrast, a possible A1 homolog in Drosophila, squid/hrp40, is, like B52, essential for proper development (Matunis et al 1992;Kelley 1993). Although it may be that squid/hrp40 is not in fact the true homolog of A1 (e.g., Haynes et al 1990), it is also possible that the protein has an essential function in development but not in cell growth.…”

Section: Discussionmentioning

confidence: 79%

Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

Wang

Takagaki²,

Manley³

1996

Genes Dev.

191

194

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Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototype of a family of nuclear proteins highly conserved throughout metazoa, the SR (serine/arginine) proteins. Based largely on in vitro studies, SR proteins have been suggested to play important roles in constitutive and alternative splicing of pre-mRNAs. Here we describe the development of a genetic system employing the chicken B-cell line DT40 to study the function of ASF/SF2 in vivo. The high level of homologous recombination and rapid growth rate of these cells allowed us to show first that ASF/SF2 is an essential gene, and then to perform targeted disruption of both ASF/SF2 alleles, by creating a cell line in which the only source of ASF/SF2 is a human cDNA controlled by a tetracycline (tet)-repressible promoter. We show that addition of tet to these cells results in rapid depletion of ASF/SF2, concomitant accumulation of incompletely processed pre-mRNA, and subsequent cell death. The tet-induced lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygous cell lines overexpressing human ASF/SF2 displayed significant reductions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels are controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processes in vertebrate cells.[Key Words: Pre-mRNA splicing; SR proteins; chicken DT40 cells] Received July 25, 1996; revised version accepted August 19, 1996.The factors involved in pre-mRNA splicing have been studied intensively in recent years. In addition to the well-characterized small nuclear ribonucleoprotein particles (snRNPs), a large number of additional factors, referred to collectively as non-snRNP proteins, are required to catalyze and/or regulate the process (for review, see Moore et al. 1993). Prominent among the nonsnRNP proteins is a family of related factors called SR proteins (Zahler et al. 1992; for review, see Fu 1995;Manley and Tacke 1996). Eight SR proteins have been described to date in mammalian systems. All contain either one or two RNP-type RNA binding domains (RBD) at their amino termini, and regions of varying lengths of arginine/serine dipeptide repeats (RS domain)at their carboxy termini. Although clear homologs to the SR proteins have not been described in yeast, the proteins are highly conserved throughout metazoa. For example, specific individual family members have been found to share ---65% identity between flies and humans (e.g., Screaton et al. 1995), and over 98% identity between chicken and humans (Vellard et al. 1992; see below).As a result of extensive in vitro studies, SR proteins have been postulated to play multiple roles in premRNA splicing. First, SR proteins are thought to be essential, or general, splicing factors, as they can reconstitute splicing of model pre-mRNAs when added to cytoplasmic S100 extracts, which lack SR proteins but contain all other essential splicing factors (Krainer et al.

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Section: Discussionmentioning

confidence: 79%

Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

Wang

Takagaki²,

Manley³

1996

Genes Dev.

191

194

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“…The RNP motif I was similar to those of chicken nucleolin (30,31), yeast nuclear localization sequence binding protein NSR 1 (32), and Drosophila hnRNP A1 (33,34) and the RNP II to those of the yeast NSR 1 (32) and tobacco chloroplast RNP precursor proteins (35) ( Table 1 and Fig. 6).…”

Section: Rnp Motif Structuresmentioning

confidence: 92%

Molecular cloning of a RNA binding protein, S1-1

Inoue

1996

Nucleic Acids Research

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S1 proteins A-D constitute a nuclear protein family that are liberated rapidly in a set from chromatin by mild digestion with a DNA or RNA hydrolyzing enzyme. With an anti-S1-protein B antiserum that reacted with B2, C1 and D1, a cDNA clone, pS1-1, was obtained, which encoded a protein of 852 amino acids. The S1-1 protein, encoded within the cells by a mRNA of 3480 nt, was a novel protein and could be distinguished from the S1 proteins B, C and D by their amino acid sequences. The S1-1 protein synthesized by in vitro translation bound to RNA homopolymers, with a preference for G and U polyribonucleotides and little for poly(A). The protein contained two tandem RNP motifs and several intriguing sequences, such as a novel repeat of five octamers with a consensus sequence DP-S(Q/G)YYY and a potentially perfect amphipathic α-helix of five turns with basic and acidic amino acids positioned in an ordered way. The two RNP motif sequences were similar, although homologies were low, to the RNP motif sequences of yeast NSR1 protein, animal nucleolins, Drosophila hnRNP A1 and tobacco chloroplast RNP precursor protein, suggesting a functional uniqueness of the S1-1 protein in RNA metabolism and also the evolution of its RNP motif structure before plants and animals diverged. These results indicate that the S1-1 protein encoded by the cDNA is a new class of RNA binding protein.

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“…The cDNAs of Hrb98DE 1A and Hrb98DE 1B, ovl2 and L3 (25), were cloned first into plasmid pBHS (32) and then into the PW8 P-element transformation vector that contains w+ as a marker (33). The resulting constructs were injected intoyw embryos with a helper plasmid that encodes transposase but is incapable of transposition itself (34 …”

Section: Methodsmentioning

confidence: 99%

“…The Drosophila HRB98DE, HRB87F, hrp36, hrp40, and hrp48 proteins and the vertebrate hnRNP A/B proteins share a common overall primary structure: two amino-terminal RNA-binding domains and a glycine-rich carboxyl-terminal domain (22)(23)(24)(25)(26)(27). However, none of these Drosophila hnRNPs display sufficient sequence similarity to any vertebrate hnRNPs to determine whether they are homologues of distinctive vertebrate hnRNPs.…”

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confidence: 99%

“…However, none of these Drosophila hnRNPs display sufficient sequence similarity to any vertebrate hnRNPs to determine whether they are homologues of distinctive vertebrate hnRNPs. The HRB98DE protein, which has 58% sequence identity with the rat Al protein in the RNA-binding domains, is probably the closest relative of Al in D. melanogaster (25). The HRB98DE gene encodes four protein isoforms derived from alternative splicing and alternative use of 5' exons 1A or 1B.…”

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confidence: 99%

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Exon skipping by overexpression of a Drosophila heterogeneous nuclear ribonucleoprotein in vivo.

Shen

Zu²,

Cass³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant RNA-binding proteins that are implicated in splicing regulation. Here we investigate the role of a Drosophila hnRNP in splicing regulation in living animals. We find that overexpression of the Drosophila hnRNP HRB98DE leads to skipping of all internal exons in the Drosophila dopa decarboxylase (Ddc) pre-mRNA in vivo. These results indicate that HRB98DE has a splicing activity that promotes use of terminal splice sites. The effect of excess HRB98DE on Ddc splicing is transient, even though high levels of HRB98DE persist for at least 24 hr. This suggests that Drosophila larvae can induce a compensating mechanism to counteract the effects of excess HRB98DE.Nascent pre-mRNAs associate with a specific group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes (1). The major protein components of hnRNP complexes from HeLa cells are the Al, A2, Bi, B2, Cl, and C2 polypeptides (2, 3). Several lines of indirect evidence suggest that hnRNPs might be involved in pre-mRNA splicing. Four hnRNPs (Al, C, D, and I/PBT) have been found to bind specifically to the conserved polypyrimidine tract located upstream of most 3' splice sites (4-6). The hnRNPs Al and I/PBT can also associate with 5' splice sites (7-9). The hnRNP Al protein has RNA-annealing activity (10-13) and can bind to the U2 small nuclear ribonucleoprotein (snRNP) (10). Efficient UV crosslinking of hnRNP Al to pre-mRNA requires both Ul and U2 snRNPs (14).Recent in vitro studies provide direct evidence that hnRNP Al might be actively involved in regulating alternative splicing. An excess of hnRNP Al promotes the use of distal 5' splice sites in model pre-mRNAs containing duplicated 5' splice sites (15). This preferential use of distal 5' splice sites can be counteracted by members of the SR protein family (16), such as SF2/ASF and SC35 (15,17,18 In this report, we investigate the role of the Drosophila hnRNP HRB98DE in splicing regulation in living larvae. We tested whether overexpression of HRB98DE affects splice site choice of the endogenous dopa decarboxylase (Ddc) premRNA (28). The Ddc pre-mRNA is expressed primarily in the central nervous system (CNS) and the hypoderm, and it is alternatively spliced in these two tissues (29). The Ddc CNSspecific mRNA contains all four exons, whereas the hypodermal mRNA contains only three exons, skipping the second exon B (see Fig. 2B) (29). Our previous results suggest that the Ddc hypodermal-specific splicing is negatively regulated by a blockage mechanism preventing splicing to exon B and indicate that the sequences of Ddc exon B and the adjacent upstream intron are sufficient for this negative regulation (30,31). Here, we show that in vivo overexpression of a Drosophila hnRNP, HRB98DE, causes profound changes in Ddc splicing. MATERIALS AND METHODSPlasmid Construction. The cDNAs of Hrb98DE 1A and Hrb98DE 1B, ovl2 and L3 (25), were cloned first into plasmid pBHS (32) and then into the PW8 P-element transformation vector that contai...

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The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes.

Cited by 53 publications

References 47 publications

Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

Molecular cloning of a RNA binding protein, S1-1

Exon skipping by overexpression of a Drosophila heterogeneous nuclear ribonucleoprotein in vivo.

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