Figure 2.

Late-scnRNAs Interact with Twi1p and Twi11p

(A) Expression of the Argonaute genes in vegetatively growing (l, m, and h indicate low, medium, and high density, respectively), starved (numbers indicate hours after removal of nutrients), and conjugating (numbers indicate hpm) cells based on a microarray analysis.

(B) Twi1p and Twi11p from wild-type cells at the indicated time points of conjugation were detected by western blotting using the indicated antibodies.

(C) Different transgenic and gene-knockout strategies. In the vegetative and early- to mid-stage conjugating cells, the parental MACs (yellow) contribute all (maternal) gene expression, whereas the new MACs, which are formed from the MIC (green), provide zygotic gene expression at late conjugation.

(D and E) Proteins from cells expressing FLAG-HA-Twi1p from the maternal TWI1 loci by MAC replacement (D) or cells expressing HA-Twi11p from the zygotic TWI11 loci by MIC replacement (E) at the indicated time points of conjugation were detected by western blotting.

(F) HA-Twi11p was immunopurified using an anti-HA antibody (α-HA-IP) from the HA-TWI11 conjugating culture at 10.5 hpm. As a negative control, wild-type cells (WT) were used for similar immunopurification. Early-scnRNAs were immunopurified with Twi1p by an anti-Twi1p antibody (α-Twi1p-IP) from the wild-type conjugating culture at 3 hpm. RNAs were separated on denaturing gels and stained using a nucleic-acid-specific dye. Arrowhead indicates scnRNAs.

(G–Q) 26- to 32-nt RNAs from the indicated immunopurified RNAs or strains were analyzed as in Figure 1B. The 300-kb window on the right is marked with a green line.

See also Figure S1.