RT-PCR confirmation of extended 5'-UTRs when adjacent Pol III binding sites are compromised. A) IGV profiles of RNA-Seq reads at the FAR3 locus in wild type and tfc6 mutant strains. Red arrow indicates extended 5'-UTR. B) Schematic diagram of the FAR3 locus showing relative location of upstream tDNA and location of primers to detect extended transcripts. Black promoter arrow indicates normal TSS, red promoter arrow represents the putative upstream TSS in the mutant backgrounds. RT-PCR results show enrichment of extended transcripts in both tfc6 and specific tDNA B-box mutants relative to wild type, while no significant differences in amplification were observed using primers within the open reading frame. C) and D) Similar analysis at the TIM21 locus, which is downstream of the tRNA pseudogene ETC9. Wild-type and tfc6 mutants are the same as in Figure 2, B-box mutants are strains DDY5118 and DDY5120 for the FAR3 locus and DDY4093-4095 for the TIM21 locus. IGV, integrative genomics viewer; TSS, transcription start site.