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Fig. 7. A domain in the N-terminal region of Csul is essential for the methylation of Sm proteins and localization of Tud. (A-C) Identification of the Tud9A1-N-binding domain in Csul. (A) Fulllength GST-Csul or derivatives were purified from bacteria and incubated with S^*Tag-Tud9A1-N. Bound S^*Tag-Tud9A1-N was detected as described in Fig. S1 of the supplementary material (upper panel). The amount of GST-Csul proteins was visualized by Coomassie Blue staining (lower panel). Amino acid numbers are given across the top. `Input' was one tenth of the S^*Tag-Tud9A1-N extract. The smallest fragment of Csul showing binding to Tud9A1-N encompasses the first 111 amino acids of Csul and may thus be distinct from the SmB binding site. (B) Delimitation of the Tud-binding domain using N-terminal truncation of Csul. Detection of S^*Tag-Tud9A1-N bound to the N-terminal truncated Csul polypeptides revealed that the sequence following amino acid residue 60 is required for Tud9A1-N binding. (C) Representation of the GST-Csul fragments used for the mapping and the summary of the binding results are indicated on the right. The identified domain of Csul necessary for Tud-binding is shown in blue. (D,E) Use of interstitial deletions in the N terminus region of Csul to determine the domain necessary for Tud localization in the nuage and sDMA methylation of SmB. (D) The Tud9A1-N-binding domain of Csul is required for Tud localization in the nuage and pole cell formation. Egg chambers and embryos from homozygous csul^RM females expressing the different transgenes were stained using anti-Tud (red; left column) and anti-Vas (red; right column) antibodies, respectively. DNA staining is in green. The deletions uncovering residues 21-40 and 41-60 can properly target Tud in the nuage and rescue the csul phenotype. (E) The N-terminal region of Csul is necessary for sDMA synthesis on SmB protein. Transgenes expressing full-length or modified Csul proteins were introduced into the csul^RM background and ovarian extracts were prepared from the homozygous females. Western blot analysis using -SYM10 antibodies indicates that none of the deletion transgenes are able to rescue the methylation of the SmB protein (arrow).

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