Fig. 1

Nbr harbors a exonuclease domain and is conserved from worms to humans. a schematic presentation of some 3′-5′ exonuclease-containing proteins, drawn to scale. From top: human Werner syndrome protein (accession number L76937.1), human FLJ371119 mRNA (accession number AK094438.1, corrected for some sequencing errors, based on genomic DNA), Drosophila melanogaster nibbler (nbr), (CG9247, accession number NM_136250.2), Caenorhabditis elegans ribonuclease-like protein ZK1098.3 (accession number NM_066703.1), Caenorhabditis elegans Mut7 protein (accession number NM_066704.1) and E. coli RNase D (accession number X07055). Identified domains are indicated on the right part and comprise the 3′-5′ exonuclease domain, the DEAD box, the Helicase C domain, the HDRC domain and a low homology region common to CG9247, human FLJ20433 and C. elegans ZK1098.3. Note that human FLJ20443 and Caenorhabditis elegans ZK1098.3 are likely nbr orthologues as they also contain a low-homology region (shaded oblique) common to all Nbr proteins. b Amino acid sequence alignment of the catalytic part of the 3′-5′ exonuclease domain of the 6 proteins in Fig. 1a. Subdomain I- III nomenclature taken from [53]. Identical amino acids appear black, conservative changes appear in grey. Critically conserved amino acids appear in bold under the alignment. The critical amino acid D of subdomain I that was exchanged to N in Fig. 2 is highlighted in red/yellow. c Exon-intron structure of nbr. Gray boxes represent exons. The line above exon 1 represents the probe used for in situ hybridization, as well as for the product of the RT-PCR for silencing quantification. The double line indicates the dsRNA fragment used for silencing in S2 cells. The hairpin line above indicates the dsRNA generated in the RNAⁱ transgenic lines. The asterisk represents the position of the aspartic acid in domain I mutated in UAS-EGFP-Nbr-N construct. d Silencing effect of transgenic RNAⁱ embryos. Agarose gel electrophoresis of the RT-PCR products amplified for 29 cycles or 32 cycles. Genotypes: UAS-nbr ⁱ 37/UAS-nbr ⁱ 37; tub-GAL4/TM3 (lanes 1 and 3); and UAS-nbr ⁱ 37/UAS-nbr ⁱ 37; +/TM3 (lanes 2 and 4). Amplification of nbr generates a 430 bp band (see Fig. 1c), compared to the 388 bp band of the internal control from ribosomal protein 49 (rp49). e Deduced amino acid sequence of the boundary of EGFP-Nbr used in the over-expression analysis of Fig. 2. Left side C terminus of EGFP (capital letters), middle linker region (small letters), right side N-terminus of Nbr (capital letters)