Fig. 5
Impact of POLR3A G672E mutation on Pol III function in human cells. a) Immonufluorescence experiment showing the predominant nuclear localization of FLAG-tagged variants of POLR3A (WT or G672E). Scale bar = 20 μm. b) FLAG-tagged variants of POLR3A (WT or G672E) were expressed at equivalent levels in HeLa cells and purified using anti-FLAG affinity chromatography. The co-purified proteins were identified by LC-MS/MS. The heatmap contains the log2-transformed average spectral count ratios of G672E/WT across both replicates. Spectral counts were computed with Mascot. Specific and shared (with Pol I and/or Pol II) subunits are identified on the left. POLR3A (the bait) is identified by an asterisk. c) ChIP-qPCR performed against FLAG-tagged variants POLR3A-WT and POLR3A-G672E expressed transiently at equivalent levels in HEK293 cells. The chromatin was quantified by qPCR with primers for two Pol III target gene promoters (VTRNA1-1 and tRNA-iMet). Pol III enrichment at these loci was calculated relative to a locus on chromosome 13 that is not bound by Pol III. Data are represented as mean +/− SEM of biological triplicates