BioProject

Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. More...

Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in the absence of Spt5, a fraction of Pol II molecules are dislodged during elongation thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15-20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed and processivity. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning towards maximum elongation speed 15-20 kb from the promoter. Overall design: For all experiments, we ablated the Supt5h gene in vitro by adding 4-hydroxy tamoxifen (4-HT) to primary Supt5hFl/- Rosa26ERT2-cre/+ mouse embryonic fibroblast (MEF) cultures for 72 hours. As controls, we used Rosa26ERT2-cre/+ primary MEFs treated with 4-HT for 72 hours (referred to as wild-type, WT). Less...