In eukaryotes, at least three Pols (I, II, and III) are responsible for synthesizing unique RNA products. Many trans-acting factors affect the efficiency of transcription by the three Pols. Some of these factors influence more than one of the nuclear Pols. One such factor is polymerase-associated factor 1 complex (Paf1C). Paf1C, composed of five subunits in Saccharomyces cerevisiae (yeast), has been shown to promote transcription by Pols I and II and is conserved across eukaryotes. Although several studies have demonstrated that Paf1C associates with Pol I machinery, its roles in ribosomal RNA synthesis are not well-defined. In this study, we used native elongating transcript sequencing (NET-seq), to investigate the effect of the loss of two of the five Paf1C subunits (Paf1 and Cdc73) on Pol I occupancy at single-nucleotide resolution in yeast. We found that in both paf1Δ and cdc73Δ mutants, there was a significant reduction in Pol I occupancy at the 5' end of the DNA template as compared to WT yeast, accompanied by other occupancy pattern changes throughout the gene. To complement these results, we also analyzed a PRO-seq dataset that was generated with DLD1 mammalian cells. Interestingly, we found that when Paf1C was knocked-down, there was also a reduction in the occupancy of Pol I at the 5' end of the gene, consistent with our NET-seq analysis. Overall, our results support the conclusion that Paf1C is an important transcription elongation factor for Pol I and may play a conserved role across species.

• PMID: 40398673
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Journal Article

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Huffines AK, Yang NJ, Schneider DA

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