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

The mechanism of assembly of RNA polymerase III (Pol III), the 17-subunit enzyme that synthesizes tRNAs, 5 S rRNA, and other small-nuclear (sn) RNAs in eukaryotes, is not clearly understood. The recent discovery of the HSP90 co-chaperone PAQosome (Particle for Arrangement of Quaternary structure) revealed a function for this machinery in the biogenesis of nuclear RNA polymerases. However, the connection between Pol III subunits and the PAQosome during the assembly process remains unexplored. Here, we report the development of a mass spectrometry-based assay that allows the characterization of Pol III assembly. This assay was used to dissect the stages of Pol III assembly, to start defining the function of the PAQosome in this process, to dissect the assembly defects driven by the leukodystrophy-causative R103H substitution in POLR3B, and to discover that riluzole, an FDA-approved drug for alleviation of ALS symptoms, partly corrects these assembly defects. Together, these results shed new light on the mechanism and regulation of human nuclear Pol III biogenesis.

Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.

Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1-GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1-GFP, τ0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1-GFP exhibits two lifetimes, τ1 = 0.95 ± 0.02 and τ2 = 2.46 ± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ± 1.5%. Also, τ and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a1 vs % holoCcp1-GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccp1-GFP with heme bound can be determined from a1. Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcp1-GFP (i.e., mature Ccp1) and 70% apoCcp1-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.