RNA binding by Sxl proteins in vitro and in vivo.

Samuels, Mark E.; Bopp, Daniel; Colvin, Richard A.; Roscigno, R F; García-Blanco, Mariano A.; Schedl, Paul

doi:10.1128/mcb.14.7.4975

Cited by 94 publications

(81 citation statements)

References 66 publications

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“…Consistent with the known sequence specificity of the Sxl protein (30,46,49,55,60), Sxl regulation requires a poly(U) run located in the polypyrimidine tract of the default 3Ј splice site. The introduction of C residues into this poly(U) run can reduce or eliminate regulation in vivo and Sxl protein binding in vitro (49,56,57). Polypyrimidine tracts in 3Ј splice sites are targets for such proteins as U2AF (64,65) that are thought to facilitate interactions between the U2 small nuclear ribonucleoprotein particle (snRNP) and the branch point of pre-mRNAs.…”

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confidence: 74%

“…In order to generate embryos homozygous for the 1621 allele, flies from the Snf 1621 Sxl M1 stock were maintained at 18ЊC and embryos were collected at 18 or 25ЊC. As indicated, extracts were centrifuged through 10 to 45% sucrose gradients as described previously (49). Gradient fractions were collected and analyzed either by trichloroacetic acid precipitation or by immunoprecipitation; in either case, analysis was followed by Western blotting (immunoblotting).…”

Section: Methodsmentioning

confidence: 99%

“…Oregon R wild-type flies were grown at 25ЊC in population cages. Typically, 0-to 15-h embryos were collected, and low-salt sonicated nuclear extracts were prepared as described previously (49). In order to generate embryos homozygous for the 1621 allele, flies from the Snf 1621 Sxl M1 stock were maintained at 18ЊC and embryos were collected at 18 or 25ЊC.…”

Section: Methodsmentioning

confidence: 99%

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Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

Deshpande

Samuels

Schedl

1996

Molecular and Cellular Biology

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The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein.In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites.In the fruit fly Drosophila melanogaster, the activity state of the binary switch gene, Sex-lethal (Sxl), is set early in development in response to the primary sex determination signal, the X chromosome-to-autosome ratio (15,17,20,32). Sxl is turned on in 2X/2A animals (females) by activating a posttranscriptional autoregulatory feedback loop (4,14). In this feedback loop, Sxl proteins promote their own synthesis by directing the female-specific splicing of Sxl transcripts originating from the Sxl maintenance promoter, Sxl-Pm (4). This autoregulatory feedback loop is responsible for maintaining the female-determined state during most of development. The Sxl autoregulatory feedback loop is not activated in 1X/2A animals (males), and Sxl-Pm transcripts are spliced in the nonproductive default mode. The critical difference between the Sxl mRNAs in the two sexes is the male-specific exon, L3 (Fig. 1). This exon contains in-frame translation stop signals that prematurely truncate an open reading frame which begins in exon L2 (5). Exon L3 is included in all male Sxl mRNAs, and these RNAs encode only very short, presumably nonfunctional polypeptides. In females, the Sxl protein mediates the skipping of the male exon, joining exon L2 to L4. This joining creates a long open frame which is predicted to encode protein species with sizes of about 35 kDa that contain two RNA recognition motif (RRM) domains (48,50).Sxl controls sexual differentiation by regulating gene cascades specifying different aspects of somatic sexual development (3, 28, 41). The best understood cascade is the tr...

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confidence: 74%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

Deshpande

Samuels

Schedl

1996

Molecular and Cellular Biology

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“…The sex-specific splicing factor Sex-lethal (SXL) regulates the splicing of transformer (tra) pre-mRNA by competing with U2AF 65 for binding to the polypyrimidine tract of the regulated 3Ј splice site. This permits the use of an alternative 3Ј splice site that is normally not selected, thus resulting in the production of a functional TRA protein only in female flies (11)(12)(13)(14)(15)(16). Direct competition with constitutive splicing factors has been shown to be a conserved strategy for vertebrate alternative 3Ј splice site choice and often involves the polypyrimidine tract binding protein (PTB/hnRNP I) (15,(17)(18)(19).…”

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confidence: 99%

Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion

Côté¹,

Dupuis²,

2001

Journal of Biological Chemistry

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We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3 portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3 acceptor site that engages in nonproductive commitment complex interactions with the 5 splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3 acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3 splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism.Pre-mRNA alternative splicing is an important mechanism for higher eucaryotes to regulate cell type-and developmental stage-specific gene expression. It provides a potential for an extraordinarily high level of diversity in generating multiple, often functionally distinct, protein isoforms from a single gene. In addition to the basic splicing signals (5Ј splice site, branch point sequence and pyrimidine tract-AG), numerous sequence elements have been identified in exons or introns that can influence in various ways the function of the splicing machinery (reviewed in Refs. 1-3). These regulatory elements can, in some cases, mediate their effects in cis, for example, through the formation of stem-loop structures (e.g. Ref. 4), although they will usually interact with trans-acting factors. Such factors often form multicomponent complexes that can contain combinations of known constitutive splicing factors, including hnRNPs, 1 snRNPs, and serine-arginine (SR) proteins as well as novel specific alternative splicing factors (e.g. Refs. 5-7). However, little is known about the precise mechanisms by which these specific complexes interact with and influence the function of the splicing machinery. Similarly, the process of splice site selection in complex pre-mRNAs is still a poorly understood phenomenon (8 -10).One mechanism for intronic elements to function in repressing splicing was first described in Drosophila. The sex-specific splicing factor Sex-lethal (SXL) regulates the splicing of transformer (tra) pre-mRNA by competing with U2AF 65 for binding to the polypyrimidine tract of the regulated 3Ј splice site. This permits the use of an alternative 3Ј splice site that is normally not selected,...

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“…In addition, there are specific splicing factors whose function is limited to the regulation of a few alternatively spliced genes such as transformer, Sex-lethal (Sxl), and doublesex (35). These proteins act through specific intronic or exonic pre-mRNA sequences to influence splice site selection and exert their effects by either recruiting or blocking components of the general splicing machinery (18,25,26,29,30,44,46,(49)(50)(51)(52). Overall, both exon and intron sequences together with their associated binding proteins can participate in constitutive and/or alternative splice site selection.…”

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The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5′ Splice Sites

Zhu

Urano

Bell

1997

Molecular and Cellular Biology

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The Sex-lethal (Sxl) early transcripts have a unique 5 exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5 splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron.Splicing reactions remove intron sequences from premRNA and join exons together with exquisite precision. These reactions are carried out within the spliceosome, using several snRNA molecules and many associated proteins. Accuracy in splicing requires that the appropriate pre-mRNA splice sites, which may be separated by vast distances, be recognized and distinguished from among many closely related sequences. Three minimum sequence elements have been identified (33)(34)(35). At the 5Ј splice site, there is a short consensus sequence that is complementary to sequences of U1 snRNA; the branch point sequence is recognized by U2 snRNA; at the 3Ј splice site, the last two nucleotides of the intron are invariantly AG (31). In addition, in higher eukaryotic cells, a polypyrimidine tract is usually located between the branch point and 3Ј splice site; it is bound by the protein U2AF, which enhances the binding of U2 snRNA to the branch point (56, 57). To some degree, the likelihood of choosing a particular splice site correlates with how closely it matches the consensus sequence. Nevertheless, these limited sequence requirements are not sufficient to explain the accuracy observed in splice site selection, as sequences matching the consensus are often found unused within introns. Additional features have also been identified as important for selection of the correct pair of 5Ј and 3Ј splice sites (5).Sequence context beyond the splice sites and branch point can influence splice site selection and splicing efficiency. For example, exon sequences have been known for some time to influence splice site selection, and several exonic purine-rich splicing enhancers have recently been characterized (12,24,28,47,53,54). Some of these enhancers have been shown to be bound by members of the SR family of proteins, which as a class appear to be involved in joining 5Ј and 3Ј splice sites across exons as well as introns (14). While SR proteins are essential for splicing in general, they can also differentially influence the choice of splice sites in a concentration-dependent ma...

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RNA binding by Sxl proteins in vitro and in vivo.

Abstract: Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of

Cited by 94 publications

References 66 publications

Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion

The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5′ Splice Sites

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