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

Shen, Juntai; Zu, Kai; Cass, Cynthia L.; Beyer, Ann L.; Hirsh, Jay

doi:10.1073/pnas.92.6.1822

Cited by 21 publications

(20 citation statements)

References 47 publications

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“…This is likely the consequence of low transfection efficiency because only Ϸ20% of cells were transfected. To our knowledge, this is the first report of effects of SR proteins on splicing regulation of an endogenous gene in mammalian cells, although an hnRNP A1 related protein has been reported to regulate dopa decarboxylase gene alternative splicing in Drosophila (18).…”

Section: Resultsmentioning

confidence: 99%

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors

Jiang

Zhang

Rao

et al. 1998

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

137

102

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The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF͞SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.Regulated alternative splicing of pre-mRNA is an important mechanism for generating functionally different proteins from the same gene. Recently, alternative splicing has been found to be involved in processing pre-mRNAs of a number of genes acting at different steps of apoptosis pathway, ranging from membrane-associated death signal receptors to members of caspase family (1-5). Most strikingly, alternative splicing of Bcl-x, Ced-4, and Ich-1 pre-mRNAs results in the formation of products that play opposite roles in apoptosis (1-3). In Bcl-x, usage of the proximal 5Ј splice site gives rise to a transcript encoding a long form of product, Bcl-xL; while selection of the distal 5Ј splice site leads to a short product, Bcl-xS. Similar to Bcl-2, Bcl-xL can inhibit cell death; whereas, Bcl-xS antagonizes the activity of Bcl-2 (1). In Ced-4 pre-mRNA splicing, alternative usage of two different 3Ј splice sites results in the formation of two transcripts, Ced-4L and Ced-4S. Overexpression of Ced-4s promotes cell death, while Ced-4L inhibits cell death (3). In the case of Ich-1, alternative splicing also generates two products, Ich-1L and Ich-1S. Ich-1L induces cell death; whereas overexpression of Ich-1S prevents cell death (2). These observations suggest a potential role of alternative splicing in regulating apoptosis.Splicing factors of the serine-arginine-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) families are emerging as important alternative splicing regulators (6-12). The SR proteins are characterized by structural and functional similarities (8-11). They contain SR sequences and RNA recognition motifs of the ribonucleoprotein type. They play important roles in both constitutive splicing and regulated alternativ...

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Section: Resultsmentioning

confidence: 99%

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors

Jiang

Zhang

Rao

et al. 1998

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

137

102

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“…Transient expression of hnRNP A1 in this mutant cell line promoted a shift toward distal 5Ј splice site selection. Overexpression of Drosophila 2ϫ RBD-Gly general hnRNP proteins, Hrb87F/ hrp36 or Hrb97DE/hrp38, transiently affected the splice site selection of the dopa decarboxylase (Ddc) pre-mRNA in Drosophila larvae (32,40). These overexpression studies provide the first glimpses of the importance of hnRNP proteins in RNA processing in vivo.…”

mentioning

confidence: 92%

Mutations in the hrp48 Gene, Which Encodes a Drosophila Heterogeneous Nuclear Ribonucleoprotein Particle Protein, Cause Lethality and Developmental Defects and Affect P-Element Third-Intron Splicing In Vivo

Hammond

Rudner

Kanaar

et al. 1997

Molecular and Cellular Biology

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The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). We have taken a genetic approach to analyzing the role of hrp48. Mutations in the hrp48 gene were identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, we found that reduced levels of hrp48 partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition.The hnRNP (heterogeneous nuclear ribonucleoprotein particle) proteins are abundant eukaryotic nuclear RNA-binding proteins that bind nascent transcripts (heterogeneous nuclear RNAs or pre-mRNAs) as they emerge from the transcriptional machinery. hnRNP complexes from vertebrates, where there are over 20 abundant, highly conserved, nuclear hnRNP proteins, designated A to U (10), have been extensively studied. These proteins are important for the packaging, processing, and intracellular transport of cellular mRNAs (5). hnRNP proteins are also found in invertebrates, such as Drosophila melanogaster. In Drosophila, several hnRNP proteins have been isolated by immunopurification and characterized by molecular cloning (20,21). There are at least 10 major hnRNP proteins in Drosophila, the most abundant being hrp36, hrp40, and hrp48. These proteins have been shown to associate with the majority of nascent transcripts (19,21).All the abundant Drosophila heterogeneous nuclear RNAbinding proteins characterized so far are similar in structure to the vertebrate A and B hnRNP proteins, containing two Nterminal RBD-type RNA-binding domains and a glycine-rich carboxyl-terminal domain (21). The RBD (or RNP-CS) type is a distinct class of RNA recognition motif (5). The RBD domains are necessary for RNA binding, and the glycine-rich region is thought to mediate RNA-binding, RNA-RNA-annealing, or protein-protein interactions (10). Proteins with these motifs are referred to as 2ϫ RBD-Gly proteins (5). Alignment of RNA-binding domains from evolutionarily diverse 2ϫ RBD-Gly proteins reveals a high degree of conservation of these domains among members of this protein family and establishes a specific consensus sequence for 2ϫ RBD-Gly proteins (5).Early evidence for a role for hnRNP proteins in pre-mRNA splicing came from antibody inhibition experiments implicating the mammalian hnRNP C protein as a splicing factor (9). More recent evidence for the role of an hnRNP protein in pre-mRNA splicing was obtained through the purification of an activity that influences alternative 5Ј splice site selection in vitro and its identification as hnRNP A1 (22). By u...

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“…In the fruit fly, there is experimental evidence for initial splice site pairing via "intron definition," since expansion of small introns leads either to their retention or to activation of a cryptic 3Ј splice site (27,62). On the other hand, several examples of exon skipping have been reported in Drosophila, both naturally occurring, as in the sex determination regulatory cascade (reviewed in reference 41) and experimentally induced (e.g., 47,57), consistent with splice site pairing via exon definition. In S. cerevisiae, only a handful of pre-mRNAs harbor more than one intron (58), and the trans-acting factors implicated in exonspanning interactions are either absent or highly divergent (1,2,8).…”

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confidence: 99%

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Romfo

Alvarez

Heeckeren

et al. 2000

Molecular and Cellular Biology

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Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with premRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5 junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5 junctions can compete effectively with the standard 5 splice site if they are closer to the 3 splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.Splice site selection has been most extensively studied in higher eukaryotes (reviewed in reference 11), where abundant evidence indicates that the unit initially recognized by the splicing machinery is the exon, as proposed by Robberson et al. nearly a decade ago (53). Particularly compelling in this regard is the observation that the most common effect of a 5Ј splice site mutation is skipping of the preceding exon rather than inclusion of the mutant intron (61; reviewed in reference 6). Moreover, in the subset of cases in which a 5Ј junction mutation causes activation of a cryptic splice site rather than exon skipping, the new exon-intron boundary is almost invariably located within the preceding exon, again supporting the view that communication occurs across the exon rather than the intron. Finally, there are significant constraints on exon length in vertebrate pre-mRNAs, consistent with the proposal that the 3Ј and 5Ј splice sites on opposite sides of the exon must be recognized concurrently. Not only are the vast majority of natural internal exons in vertebrate pre-mRNAs Ͻ300 nucleotides in length (6), but expanding an exon beyond this size causes it to be skipped (53), particularly if it is surrounded by large introns (60). In contrast to the limitations on exon length, the introns in vertebrate pre-mRNAs can be extremely large (tens of kilobases [29]).Although many questions remain to be answered, several components of the machinery responsible for exon definition have been identified. First, UV cross-linking experiments ...

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

Cited by 21 publications

References 47 publications

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors

Mutations in the hrp48 Gene, Which Encodes a Drosophila Heterogeneous Nuclear Ribonucleoprotein Particle Protein, Cause Lethality and Developmental Defects and Affect P-Element Third-Intron Splicing In Vivo

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

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