Kep1 interacts genetically with dredd/caspase-8, and kep1 mutants alter the balance of dredd isoforms

Abstract

The Drosophila kep1 gene encodes an RNA binding protein related to the murine QUAKING apoptotic inducer. We have previously shown that kep1 can induce apoptosis when transfected into different cell lines. To better define the role of Kep1 in apoptosis, we generated kep1 null flies. These flies were viable, but females displayed reduced fertility, with approximately half of the eggs laid from kep1- homozygotes failing to hatch. In addition, loss of kep1 suppressed GMR-rpr-mediated apoptosis in the Drosophila eye, and kep1 mutant flies had increased susceptibility to Escherichia coli infection. We found that Kep1 bound dredd RNA in vitro, and that extracts prepared from kep1 mutant ovaries had markedly reduced proteolytic cleavage activity toward the caspase-8 target substrate IETD-7-amino-4-trifluoromethyl coumarin. We observed increased levels of the beta isoform of dredd mRNA in kep1 mutants as compared with wild-type. Taken together, our results suggest that Kep1 regulates apoptosis by influencing the processing of dredd RNA.

Figure 1

Characterization of kep1− mutants. (A) Schematic diagram of the arrangement of kep1 and two neighboring genes in region 58E of chromosome 2 in wild-type (Top) and in the kep1− deletion (Middle). Note that much of the kep1 gene is deleted in the kep1− deletion. The wild-type and kep1− genomic sequences are given at the Bottom. PCR was used to amplify the region surrounding the P-element excision site in kep1− homozygotes, the amplified fragment was sequenced, and its sequence was compared with that of a BAC clone that did not contain the original P element (BACR48M13). The start of the kep1 cDNA in BACR48M13 is at position 89990. The sequence of the remaining fragment of the P element, after the imprecise excision event, is underlined. (B) Phenotypes of eggs laid by kep1− mutant females; details and frequencies of the different phenotypes are described in Table 1.

Figure 2

Kep1 protein can be UV crosslinked to dredd mRNA. (A) Radiolabeled RNAs (refer to Methods) were incubated in the presence of HeLa cell extracts transfected with either myc-Sam50 (lanes 1, 2, 5, 6, 9, and 10) or myc-kep1 (lanes 3, 4, 7, 8, 11, and 12). These extracts were immunoprecipitated with control serum (odd-numbered lanes) or 9E10 anti-Myc antiserum (even-numbered lanes). (B) Myc immunoblot using 9E10 anti-Myc antiserum showing the presence of the transfected proteins in both extracts. (C) Organization of part of the dredd gene. Exons are shown as dark boxes; arrows indicate the relative position of the oligonucleotides used in the RT-PCR experiments to investigate the nature of the different dredd mRNA isoforms in various mutants. (D) RT-PCR were fractionated on a 1.5% agarose gel. Lanes indicate the source of the mRNA used in the reverse transcription reactions in the presence (+RT) or absence of reverse transcriptase.

Figure 3

IETDase activity in ovary extracts. Extracts were prepared and IETDase activity was measured as described in Methods. Results are the average readings from duplicate samples from one representative experiment. RFU, relative fluorescent units. OreR was used as the wild-type control, and other extracts were analyzed from kep1− heterozygotes (kep1−/CyO) and homozygotes (kep1−/−), homozygotes for two alleles of dredd (EP1412/EP1412, B118/B118), and ovaries obtained from flies carrying two copies of the kep1 transgene in a kep1 mutant homozygous background (P[kep1];kep1−/−).

Figure 4

Susceptibility of flies to E. coli infection. Flies were challenged with E. coli or subjected to a mock infection as described in Methods. OreR was used as the wild-type control, and survival was also scored for kep1− heterozygotes (kep1−/CyO) and homozygotes (kep1−/−), and homozygotes for two alleles of dredd (EP1412/EP1412, B118/B118).

Figure 5

Scanning electron micrographs of compound eyes. Flies of various genotypes were visualized by scanning electron micrographs as described in Methods. (A) yw. (B) kep1−/kep1−. (C) B118/B118. (D) GMR-rpr/+. (E) kep1−/kep1−;GMR-rpr/+. (F) B118/B118;GMR-rpr/+. The arrow illustrates an example of cell overgrowth occasionally seen in kep1−/kep1− mutant eyes.