Abstract
We describe here the identification and functional characterization of the enzyme O-GlcNAcase (OGA) as an RNA polymerase II elongation factor. Using in vitro transcription elongation assays, we show that OGA activity is required for elongation in a crude nuclear extract system, whereas in a purified system devoid of OGA the addition of rOGA inhibited elongation. Furthermore, OGA is physically associated with the known RNA polymerase II (pol II) pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1β, and a purified OGA-SPT5-TIF1β complex has elongation properties. Lastly, ChIP-seq experiments show that OGA maps to the transcriptional start site/5′ ends of genes, showing considerable overlap with RNA pol II, SPT5, TRIM28-KAP1-TIF1β, and O-GlcNAc itself. These data all point to OGA as a component of the RNA pol II elongation machinery regulating elongation genome-wide. Our results add a novel and unexpected dimension to the regulation of elongation by the insertion of O-GlcNAc cycling into the pol II elongation regulatory dynamics.
Highlights
The OGA requirement may impinge on the phosphorylation state of RNA pol II, as we have shown that both serine 2 and serine 5 residues of the pol II CTD can be O-GlcNAcylated [44]
Our results have described a novel regulatory space that regulates RNA pol II transcription at promoters and during elongation
These studies connect the nutrient state of the cell to the transcriptional machinery and all that is implied by the metabolic dysregulation seen in human disease, from diabetes to cancer [73]
Summary
UDP-GlcNAc levels represent a nutrient sensor and reflect the intracellular glucose concentration [35]. We had previously found requirements for both OGT and OGA in transcription in vitro [43], the point at which the enzymes acted was not determined. We examined OGA function further using in vitro transcription assays. In vitro elongation assays showed a requirement for OGA during elongation in crude nuclear extracts. We identified an ϳ600-kDa complex that contains OGA and the pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1 [27, 28]. In vivo ChIP-seq analysis shows that OGA localizes to TSS and OGA peaks overlap considerably with pol II, SPT5, and TRIM28-KAP1-TIF1 peaks. These data suggest a novel role for OGA activity in the regulation of RNA pol II pausing and elongation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
