Distributive O-GlcNAcylation on the Highly Repetitive C-Terminal Domain of RNA Polymerase II

Abstract

O-GlcNAcylation is a nutrient-responsive glycosylation that plays a pivotal role in transcriptional regulation. Human RNA polymerase II (Pol II) is extensively modified by O-linked N-acetylglucosamine (O-GlcNAc) on its unique C-terminal domain (CTD), which consists of 52 heptad repeats. One approach to understanding the function of glycosylated Pol II is to determine the mechanism of dynamic O-GlcNAcylation on the CTD. Here, we discovered that the Pol II CTD can be extensively O-GlcNAcylated in vitro and in cells. Efficient glycosylation requires a minimum of 20 heptad repeats of the CTD and more than half of the N-terminal domain of O-GlcNAc transferase (OGT). Under conditions of saturated sugar donor, we monitored the attachment of more than 20 residues of O-GlcNAc to the full-length CTD. Surprisingly, glycosylation on the periodic CTD follows a distributive mechanism, resulting in highly heterogeneous glycoforms. Our data suggest that initial O-GlcNAcylation can take place either on the proximal or on the distal region of the CTD, and subsequent glycosylation occurs similarly over the entire CTD with nonuniform distributions. Moreover, removal of O-GlcNAc from glycosylated CTD is also distributive and is independent of O-GlcNAcylation level. Our results suggest that O-GlcNAc cycling enzymes can employ a similar mechanism to react with other protein substrates on multiple sites. Distributive O-GlcNAcylation on Pol II provides another regulatory mechanism of transcription in response to fluctuating cellular conditions.

Interplay between Metabolism and Epigenetics: A Nuclear Adaptation to Environmental Changes

“…In addition to phosphorylation by various cyclin-dependent kinases (CDK7 of the TFIIH complex, CDK8, CDK9, CDK12 and CDK13) (reviewed in Buratowsky 2009 Egloff et al, 2012), the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) is also O-GlcNAcylated (Figure 2). More specifically, serine 5 of CTD is targeted by a dual competition between phosphorylation and O-GlcNAcylation (Kelly et al, 1993; Comer et al, 2001; Ranuncolo et al, 2012; Lu et al, 2016). Because phosphorylated CTD at serine 5 is a prerequisite for Pol II transcriptional pausing, it is tempting to speculate that O-GlcNAcylation of this serine residue might regulate the formation of the paused Pol II transcriptional complex.…”

Crosstalk between phosphorylation and O‐GlcNAcylation: friend or foe

“…Thus, efforts more recently have turned to search for a reciprocal O-GlcNAcylation/ phosphorylation crosstalk motif. This O-GlcNAcylation on RNA polymerase, however, only occurs efficiently when more than~10 heptad repeats are present [128,129], thus factors in addition to sequence alone likely govern which proteins can undergo reciprocal O-GlcNAcylation/phosphorylation crosstalk. Interestingly, proteins containing these motifs were enriched in specific gene ontology terms such as nuclear transport, cytoskeleton and structure molecular activity, suggesting specific biological functions for PTM crosstalk [126].…”

“…In addition, O-GlcNAcylation also occurs on the heptad repeats (YSPTSPS) in the C-terminal domain of RNA polymerase. This O-GlcNAcylation on RNA polymerase, however, only occurs efficiently when more than~10 heptad repeats are present [128,129], thus factors in addition to sequence alone likely govern which proteins can undergo reciprocal O-GlcNAcylation/phosphorylation crosstalk.…”

Semi-processive hyperglycosylation of adhesin by bacterial proteinN-glycosyltransferases

“…A distributive mechanism has been observed for the OGT-catalyzed O-GlcNAcylation of RNA polymerase II. 23 Processivity is a challenging trait to study, and established methods have been reviewed elsewhere. 22,24 We selected HiNGT (R2846_0712) and its close homolog ApNGT (APL_1635, 65% identity and 85% similarity) 12 and using the C-terminal region of the natural HMW1A adhesin (HMW1ct, from H. influenzae, Figure S1) as acceptor substrate, we show that both NGTs display semi-processive behaviour ( Figure 1C).…”