Drosophila SPF45: a bifunctional protein with roles in both splicing and DNA repair

Abstract

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair.

Figure 1. D. melanogaster SPF45 Protein Is Conserved

Alignment of SPF45 proteins from D. melanogaster and humans and the DRT111 protein from A. thaliana. Identical residues are shaded. The residues that comprise the G-patch domain are shaded in red, and the residues that comprise the RRM-like motif are shaded in blue. An arrow indicates the position of the P-element insertion in spf45^J23.

Figure 2. Association between SPF45 and the U2 nsRNP Is Mediated by a Direct Interaction between the C-Terminal End of SPF45 and U2A′

(A) Diagram of the 403–amino-acid SPF45 protein. An arrow indicates the position of the P-element insertion in spf45^J23. (B) Western blots of U2A′, SPF45, and SNF immunoprecipitations (IP) from nuclear extracts showing that SPF45 can be co-immunoprecipitated with SNF and U2A′. Previous studies have shown that SNF and U2A′ directly interact [26] and are included here as positive controls. (C) Western blot of extracts made from wild-type and spf45 mutant animals probed with a polyclonal antibody against SPF45 shows that the expected size band is replaced by an ∼37 kD band in spf45^J23. In extracts made from adults (but not in nuclear extracts made from embryos), the SPF45 antibody picks up an additional higher molecular weight cross-reacting band of unknown origin, which can serve as a loading control. (D) Yeast two-hybrid interactions showing that SPF45 interacts with U2A′, and that this interaction is mediated by the RRM domain. Positive interactions were tested by assaying the ability of the transformed yeast to grow on selective media after 3 days. (E) Western blots of U2A′ and SPF45 immunoprecipitations (IP) from wild-type and mutant lysates showing that the SPF45^J23 does not co-immnoprecipitate with SNF in mutant extracts, whereas the control splicing proteins, SNF and U2A′, are co-precipitated.

Figure 3. Sxl Splicing Is Compromised in a spf45 Loss-of-Function Background

(A) Synergistic genetic interactions between Sxl and spf45^J23 leads to female lethality. In these assays females of the indicated genotype were mated to either Sxl^f1/Y males or Sxl⁺/Y control males and the resulting progeny scored. On the assumption that an equal number of male and female progeny should be generated from each cross, the percent female viability was calculated by comparing the number of females recovered with the number of males recovered. (B) Diagram of the Sxl reporter construct that mirrors native Sxl splicing in all tissues tested. The arrows below the construct indicate the position of the PCR pairs used for RT–PCR. (C) Synergistic lethal interactions between Sxl and spf45^J23 are correlated with Sxl splicing defects. Splicing was assayed by an RT–PCR-based assay using RNA isolated from a pool of embryos in which only the female embryos carry the reporter construct (lanes 3 and 4). In lane 4, this pool of embryos was collected from spf45^J23 homozygous females crossed to males carrying an X chromosome that carries both Sxl^f1 and a copy of the Sxl reporter construct. Controls include splicing of the reporter construct in adult males (lane 1), splicing of the reporter construct in adult females (lane 2), and Sxl⁺ embryos collected from spf45^J23 homozygous mothers (lane 3).

Figure 4. SPF45 Function Is Required to Repair DNA Damage

(A) Ectopic expression of SPF45 improves the survival rate of E. coli recG mutants after MMS exposure. The survival was expressed as the average-fold increase in survival over the control E. coli recG mutants. Error bars are standard deviations calculated from five independent experiments. An unpaired t test was performed to assess whether there is a statistically significant difference between survival rates of an E. coli recG strain expressing a D. melanogaster protein (either SPF45^J23 or SPF45⁺) and the survival rate of the same E. coli recG strain expressing an empty GST vector. Asterisks denote differences significant at the 95% level (p < 0.04). (B) spf45 animals have an MMS-sensitive mutant phenotype. Survival was assessed by comparing the number of mutant animals that survived to adulthood with the number of control siblings (spf45^J23/CyO or Df(2Lh)D1/CyO) recovered from the same cross. (C) Damage after P-element excision is not repaired efficiently in an spf45 mutant background. Eye colors of female progeny that inherited the repaired P{w^a} were scored. The total number of females scored is indicated below the relevant genotype. A chi square test was performed to assess whether there is a significant difference between the frequency of repair events. Asterisks denote differences significant at the 99.9% level (p < 0.0005).

Figure 5. Genetic and Physical Interactions between SPF45 and RAD201, a Member of the RecA/Rad51 Protein Family

(A) spf45^J23 does not complement the MMS mutant phenotype of rad201. Relative survival to adulthood of the double heterozygous mutant animals was assessed by comparing the number of experimental animals with the number of sibling controls. (B) rad201 splicing in wild-type and spf45^J23 mutants. Genomic organization of rad201 (CG2412). The position of the C-to-T transition leading to a proline-to-leucine change at position 95 in the rad201¹ open reading frame is indicated. The rad201 gene encodes a single transcript. RT–PCR analysis using primers that span the first and second introns demonstrates that splicing is unaffected in spf45^J23 mutants. (C) RAD201 interacts with SPF45 in whole-cell extracts in an RNA-independent manner. The ability of RAD201 to associate with SPF45 in whole-cell extracts was tested by GST pull-down assays followed by Western blotting. GST::RAD201, GST::U2A′, or GST alone, bound to glutathione sepharose beads, were incubated with extracts made from wild-type or spf45^J23 embryos, followed by Western blotting using an antibody directed against SPF45 or SNF. The lane marked 5% input is a control in which the amount of extract corresponds to ∼5% of the material applied to the glutathione affinity beads. The RNase sensitivity of these interactions was tested by pretreating the extracts with a combination of RNase A and RNase T1.