FIG. 6.

Spf30, recruited to centromeric heterochromatin in a Dis3-dependent manner, binds nascent centromeric transcripts. (A) The chromosomal association of Dis3 is unaffected in spf30-38 cells. Dis3 levels at indicated chromosomal sites were assessed by ChIP. The indicated values correspond to averages and m.d. calculated from four (wild type) and six (spf30-38) experiments. (B) Spf30 colocalizes with Dis3 at centromeres and euchromatic genes and is reduced at the pericentric heterochromatin in dis3-54 cells. Spf30 binding was assessed by ChIP. The indicated values correspond to averages and m.d. calculated from four experiments. (C) Spf30 binds RNA species, among which were centromeric transcripts. The indicated strains were processed for RNA-IP by using polyclonal anti-GFP antibodies. RNA recovered in the input and immunoprecipitated fractions was reverse transcribed in the presence (+) or absence (−) of reverse transcriptase. cDNAs were quantified by real-time PCR. The indicated values correspond to averages and m.d. calculated from two independent experiments with two reverse transcriptions per experiment. (D) Spf30 associates with chromatin in an RNA-dependent manner. Chromatin extracts prepared for ChIP were treated with RNase or RNase plus inhibitor before immunoprecipitation. Enrichments in Spf30-GFP immunoprecipitated fractions were calculated as a ratio, i.e., the quantity in the immunoprecipitate/the quantity in the input, normalized to the ratio of the corresponding untagged control. The indicated values correspond to averages and m.d. calculated from two experiments.