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doi: 10.1242/10.1242/dev.00274

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Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping

Alexis A. Nagengast1, Shane M. Stitzinger1, Chin-Hsiu Tseng2,*, Stephen M. Mount2 and Helen K. Salz1,{dagger}

1 Department of Genetics, Case Western Reserve University, Cleveland, OH 44106-4955, USA
2 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
* Present address: Invitrogen Corporation, Carlsbad, CA 92008, USA



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  		Fig. 3. Analysis of the snf148 mutant phenotype. (A) Wild-type ovarioles stained with the nuclear dye DAPI. (B) Homozygous snf148 mutant ovaries stained with DAPI contain tumorous egg chambers that are filled with large numbers of undifferentiated cells. (C) Wild-type ovarioles stained with an antibody directed against SNF, illustrating that SNF localizes to the nucleus. (D) Homozygous snf148 mutant ovaries stained with an antibody directed against SNF. The magnification of a single mutant egg chamber (insert) illustrates that this mutation does not alter the nuclear localization of SNF. (E) Diagram of the reporter construct that mimics Sxl splicing in all tissues. The arrows below the construct show the positions of the nested PCR primer sets used for RT-PCR. (F) To analyze the RNAs produced by the Sxl reporter construct, total RNA was isolated from either adults or isolated ovaries of the indicated genotype, and analyzed by semi-quantitative RT-PCR.

 


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  		Fig. 4. SXL associates with the U1 snRNP particle in embryonic extracts. (A) The SXL/U1-70K association is more robust than the SXL/SNF association. The ability of SXL to associate with SNF and U1-70K in whole cell extracts was tested by GST pull-down assays, followed by western blotting. The RNase sensitivity of these interactions was tested by pre-treating the embryonic extracts with a combination of RNaseA and RNase T1. (B) SXL associates with U1-70K in snf148 mutant extracts. GST pull-down experiments were carried out as in Fig. 2, with extracts made from wild-type and a homogenous population of embryos whose only source of SNF protein is the mutant SNF148 protein.

 


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  		Fig. 1. Impact of snf mutations on snRNP assembly. (A) Schematic representation of SNF, the Drosophila U1A/U2B'' protein. SNF contains two RRM domains (for RNA recognition motif) separated by a short linker region. The arrows indicate the location of the mutations used in this study. As indicated, the sequence of the N- and C-terminal RRM motifs share significant sequence identity with both the human U1A and U2B'' proteins. (B) Amino acid sequence alignment of the N-terminal RRM domain from SNF and the human U1A and U2B'' proteins and the amino acid substitutions associated with each snf allele used. Identical amino acids are indicated by black dots above the sequence. (C) snRNP incorporation was tested by immunoprecipitation of SNF from extracts made from adult flies of the indicated genotype followed by northern blotting to detect U1 and U2 snRNAs in RNA extracted from the precipitated fractions.

 


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  		Fig. 2. Impact of snf mutations on SXL-SNF complex formation. SXL/SNF complex assembly was tested by GST pull-down assays. Equal amounts of GST::SXL fusion protein, or GST alone, bound to glutathione sepharose beads were incubated with embryonic extracts of the indicated genotype followed by western blotting using an antibody directed against SNF. Because a substantial amount of maternally produced SNF protein is supplied to the embryo, we carried out crosses (described in the Materials and Methods) to obtain a homogeneous population of embryos whose only source of SNF protein (both maternal and zygotic) is the mutant protein. The lane marked 10% input, is a control in which the amount of snf148 extract loaded corresponds to ~ 10% of the material applied to the glutathione affinity beads.

 


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  		Fig. 5. Mutations in the genes encoding the U2AF38 and U1-70K splicing factors are dosage-sensitive maternal modifiers of Sxl splicing autoregulation. (A) Synergistic genetic interactions between splicing factors leads to female-lethality. In these assays females of the indicated maternal genotype were mated to Sxl7BO/Y males and the resulting male and female progeny scored. The viability of the female progeny, all of which are heterozygous for Sxl (Sxl7B0/+), was assessed by comparing the number of females recovered to the number of males recovered. (B) Sxl splicing pattern in Sxl7B0/+ female embryos. Splicing was assayed by an RT-PCR based assay, in which RNA was isolated from a pool of embryos in which only the Sxl7B0/+ embryos carried the reporter construct. This pool of embryos was collected from the experimental adult females crossed to males carrying an X-chromosome which carries both Sxl7B0 and a copy of the Sxl reporter construct described in Fig. 3E. Lanes 3-5: embryos were collected from snfJ210/+ control mothers (lane 3); snfJ210/+; U2af38{Delta}E18/+ mothers (lane 4); and snfJ210/+; U1-70K1/+ mothers (lane 5). Controls include: lane 1, splicing of the reporter construct in adult males; and lane 2, splicing of the reporter construct in adult females.

 


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  		Fig. 6. SXL associates with both U2AF subunits in embryonic extracts. GST pull-down experiments were carried out as in Fig. 2, with extracts made from wild-type and a homogenous population of embryos whose only source of SNF protein is the mutant SNF148 protein.

 


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  		Fig. 7. How SXL promotes exon-skipping. In females, the SXL protein (pink ovals) binds to several sites within the introns located both upstream and downstream of the male-exon (blue rectangle), and associates with both U2AF and the U1 snRNP to block the 3' and 5' splice sites from being used. Formation of this dead-end complex guarantees that the male-exon will be skipped, and that exon 2 is spliced to exon 4. In males, where there is no SXL protein, U2AF and the U1 snRNP are free to assemble into an active spliceosome and exon 3 is included in the mature transcript. AG indicates the location of the two male exon 3' splice sites.

 

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