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Fig. 2 | Molecular Brain

Fig. 2

From: Riluzole partially restores RNA polymerase III complex assembly in cells expressing the leukodystrophy-causative variant POLR3B R103H

Fig. 2

Time-course of Pol III assembly as determined by sequential AP-MS experiments using FLAG-POLR3A.

FLAG-tagged POLR3A WT was transfected in HEK293 in triplicate and cells were harvested at time points indicated in the flow chart (A). Affinity purification was performed on 1.5 mg of cell extract for each time-point, dried, trypsin digested and quantified by LC-MS/MS. The LFQ intensity of each protein was computed using MaxQuant (version 1.6.17.0) against characterized Uniprot database (updated on June 3th 2018) and further analyzed by Perseus (Version 1.6.14.0) and GraphPad Prism 8 (Version 8.4.3). Proteins found in all triplicates were kept and Missing Not At Random (MNAR) values were imputed. Shown in (B) are histograms of indicated proteins significantly associated with POLR3A WT (n = 3). Proteins were deemed significantly associated when they had a False Discovery Rate (FDR) of p < 0.05 obtained via a two-tailed t-test adjusted with a permutation-based multiple hypothesis testing with 10,000 iterations and an s0 correction factor of 0.1 and the log2-transformed average LFQ-intensity difference between FLAG-Tagged POLR3A and the FLAG control ≥ 1. C) The model summarizes the multistep formation of the POLR3A subcomplex, following interaction between POLR3A subunit and 7 other Pol III subunits. Asterisks mark the subunits used as baits

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