Differential effect of the Pol III inhibitor ML-60218 in the transcription of distinct ncRNAs

GIMENEZ, MACARENA; FERNÁNDEZ ALVAREZ, ANA JULIA; BOCCACCIO, GRACIELA L

Congreso; Riboclub 22th anual meeting; 2021

Pol III is the largest RNApolymerase complex and transcribes non-coding RNAs. Two paralogs for one of thePol III subunits, termed POLR3G and POLR3GL, exist in mammalian cells. Inproliferating cells POLR3G and POLR3GL are believed to be redundant and whenboth POLR3G and POLR3GL are available, Pol III can incorporate either of thesesubunits to transcribe themajority of Pol III genes (Pietri et al., 2019). POLR3G but notPOLR3GL is inhibited by ML-60218, a poorly described Pol III inhibitor. Uponexposure of mammalian cells to ML-60218, POLR3G is downregulated and POLR3GLshows increased association with the core Pol III subunit (Pietri et al., 2019). More recently,mapping studies showed that ML-60218 disrupts the occupancy of both POLR3G andPOLR3GL in opposite manner. POLR3G presence is reduced in most PolIII-transcribed genes whereas POLR3GL occupancy is largely unaffected, or evenincreased in anumber of genes (Van Bortle et al., BioRxiv 2021). Based on theseobservations, we speculate that transcripts that depends on POLR3G and thatcannot be transcribed by POLR3GL will be downregulated by ML-60218. Genes thatcan be transcribed either by POLR3G or POLR3GL would show a moderate or nulleffect, and transcripts that are transcribed mostly by POLR3GL would show no effect, or would be upregulated by ML-60218.Here we show that ML-60218 elicits differential effects on Vault RNAs, Y4 andU6 RNAs. None of these ncRNAs showed reduced levels upon POLR3G inhibition. Incontrast, Vault 1-2 and Y4 levels increased 4X after exposure to ML-60218. U6and Vault 1-3 were not affected and Vault 1-1 levels moderately increased.These observations are compatible with a differential dependence of POLR3G andPOLR3GL for the transcription of Y4 and Vault 1-2 in comparison with U6 andVault 1-3 RNA. The immediate upregulation of Y4 and Vault 1-2 RNAs uponexposure to ML-60218 suggests that their promoters are actively transcribed byPOLR3GL. We propose that the response to ML-60218 indirectly informs on thebalance POLR3G/POLR3GL in the transcription of a given POL III gene.