Fig. 5. Rixosome-associated RNA degradation is required for repression of Polycomb-regulated genes.
a, Schematic of rixosome RNA-processing activities depicting the hypothesis that the LAS1L endonuclease and RNA NOL9 kinase activities are required to prepare target RNA for XRN2-mediated degradation. b–d, Quantitative PCR with reverse transcription (RT–qPCR) analysis of expression of the indicated Polycomb target genes in the indicated siRNA-treated HEK 293FT cells rescued with LAS1L (b), NOL9 (c) or XRN2 (d). Expression levels were normalized to ACTB and siCtrl-treated cells. Data are presented as mean ± s.e.m. for three biological replicates. e, Schematic showing construction of cell lines with a 5×tetO-H2B-CITRINE (H2B-CTRN) reporter gene expressing rTetR–RING1B wild type or rTetR–RING1B-2A fusion proteins. f–h, ChIP–qPCR analysis of H2B-CTRN reporter enrichment for RING1B (f), H2AK119ub1 (g) and TEX10 (h) following 21 days of doxycycline treatment. ChIP–qPCR signals were normalized to GAPDH. Dots represent individual biological replicates. i, RT–qPCR analysis showing H2B-CTRN reporter RNA expression in rTetR–RING1B-expressing wild-type or RING1B-2A HEK 293FT cells before treatment relative to after 21 days of doxycycline treatment. RNA expression levels were normalized to ACTB. Data are presented as mean ± s.e.m. for three biological replicates. j, RT–qPCR analysis of H2B-CTRN RNA expression in the indicated siRNA-treated and NOL9-rescued HEK 293FT cells 3 days after release from 21-day doxcyline treatment. Expression levels were normalized to ACTB and to siCtrl-treated cells. Data are presented as mean ± s.e.m. for three biological replicates k, Model for the role of rixosome in Polycomb-mediated gene silencing. The rixosome is recruited through the interaction of RING1B in the vPRC1 and PRC1 complexes with the TEX10 subunit of the rixosome (individual subunits not shown) to mediate nascent RNA degradation and transcription termination. The rixosome also interacts with PRC2. See text for details.