Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

Abstract

Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of aminoterminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1-or mutation of ubiquitylation sites within Rpb1-induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.ubiquitin | chromatin | gene silencing | transcription T he ubiquitin (Ub)-proteasome system features in a multitude of cellular processes, including the regulation of transcription (1). Over the past two decades, it has become clear that Ubdependent processes impact multiple steps in gene expression, ranging from signaling histone modifications (2) to coordinating events of mRNA export from the nucleus (3). Many of the functions of Ub in transcription are directed against regulatory factors, and there are only a handful of examples in which core components of the transcriptional machinery are regulated by Ub-dependent transactions. The best understood example from this category is the Ub-mediated destruction of the largest subunit of RNA polymerase II (pol II) in response to DNA damage (4), which functions as a failsafe mechanism to remove stalled pol II complexes from damaged DNA segments. Beyond a damage response, however, it is unclear whether ubiquitylation controls the normal activity of pol II.We previously reported that the Saccharomyces cerevisiae protein Asr1 is a RING (really interesting new gene) finger Ub-ligase that targets RNA polymerase II for nonproteolytic ubiquitylation (5). Asr1 defines the evolutionarily preserved RPC (RING, PHD, CBD) family of Ub-ligases, characterized by the presence of amino-terminal RING and PHD (plant homeo domain) fingers an...

Ccr4–Not maintains genomic integrity by controlling the ubiquitylation and degradation of arrested RNAPII

“…The process is more complicated than anticipated, and many factors involved in degrading RNA polymerase have not been identified. For example, there is evidence that Asr1 and an unidentified ubiquitin ligase modify Rpb1 at telomeres and during DNA damage-independent arrest, respectively (Daulny et al 2008;Karakasili et al 2014;McCann et al 2016). Moreover, what distinguishes terminally arrested from paused RNAPII and what triggers the binding of the ubiquitin conjugation machinery are not known.…”

The deubiquitylase Ubp15 couples transcription to mRNA export

“…Interestingly, the histone chaperone and elongation factor FACT (Spt16 and Pob3) is also enriched in RNAPII purified from fcp1-1 cells ( Figure 1B , Figure 1—figure supplement 1B , Supplementary file 1 ) and both FACT and Rpb1 are substrates of Ubp15 in Schizosaccharomyces pombe ( Beckley et al, 2015 ). In addition, the association of Asr1, a ubiquitin ligase known to bind the CTD and to ubiquitylate RNAPII in the context of elongation ( Daulny et al, 2008 ; McCann et al, 2016 ), is decreased in fcp1-1 cells ( Figure 1B , Supplementary file 1 ). These results prompted us to investigate the possible role for Ubp15 in transcription elongation.…”

“…RNAPII was also shown to be ubiquitylated in vivo by the ubiquitin ligase Asr1 ( Daulny et al, 2008 ). In this case, the ubiquitylation occurs on transcribed genes and leads to the dissociation of two RNAPII subunits (Rpb4/7), a process involved in the silencing of subtelomeric genes ( McCann et al, 2016 ). Ubiquitylation is also involved in mRNA export ( Babour et al, 2012 ).…”

The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

“…pombe (Beckley et al ., 2015). In addition, the association of Asr1, a ubiquitin ligase known to bind the CTD and to ubiquitylate RNAPII in the context of elongation (Daulny et al ., 2008, McCann et al ., 2016), is decreased in fcp1-1 cells ( Figure 1B, Supplementary file 1 ). These results prompted us to investigate a possible role for Ubp15 in transcription elongation.…”