Communication between Distant Sites in RNA Polymerase II through Ubiquitylation Factors and the Polymerase CTD

Somesh, Baggavalli P; Sigurdsson, Stefan; Saeki, Hideaki; Erdjument‐Bromage, Hediye; Tempst, Paul; Svejstrup, Jesper Q.

doi:10.1016/j.cell.2007.01.046

Cited by 72 publications

(70 citation statements)

References 28 publications

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“…Depending on the experimental conditions used, Rsp5 may predominantly add mono-ubiquitin or a polyubiquitin chain to RNAPII in vitro (6,12). Therefore, it is formally possible that in vivo, Rsp5 normally only adds mono-ubiquitin.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, degradation of RNAPII may be a ''last resort,'' used to clear active genes of persistently arrested RNAPII elongation complexes (6-9). Interestingly, the proteasome is nuclear and can be found on the coding region of genes by chromatin-immunoprecipitation (10), so RNAPII proteolysis may well occur on the DNA.We have reconstituted RNAPII ubiquitylation in vitro with highly purified, physiologically relevant yeast, or human, ubiquitylation factors, respectively (6,11,12). The yeast HECT E3 Rsp5 binds RNAPII via the flexible C-terminal repeat domain (CTD) of the Rpb1 subunit (13), but modifies the polymerase in the main body of the Rpb1 subunit (6,14).…”

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confidence: 99%

“…We have reconstituted RNAPII ubiquitylation in vitro with highly purified, physiologically relevant yeast, or human, ubiquitylation factors, respectively (6,11,12). The yeast HECT E3 Rsp5 binds RNAPII via the flexible C-terminal repeat domain (CTD) of the Rpb1 subunit (13), but modifies the polymerase in the main body of the Rpb1 subunit (6,14).…”

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Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation

Harreman

Täschner²,

Sigurdsson³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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152

143

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The proteasome degrades proteins modified by polyubiquitylation, so correctly controlled ubiquitylation is crucial to avoid unscheduled proteolysis of essential proteins. The mechanism regulating proteolysis of RNAPII has been controversial since two distinct ubiquitin ligases (E3s), Rsp5 (and its human homologue NEDD4) and Elongin-Cullin complex, have both been shown to be required for its DNA-damage-induced polyubiquitylation. Here we show that these E3s work sequentially in a two-step mechanism. First, Rsp5 adds mono-ubiquitin, or sometimes a ubiquitin chain linked via ubiquitin lysine 63 that does not trigger proteolysis. When produced, the K63 chain can be trimmed to mono-ubiquitylation by an Rsp5-associated ubiquitin protease, Ubp2. Based on this mono-ubiquitin moiety on RNAPII, an Elc1/Cul3 complex then produces a ubiquitin chain linked via lysine 48, which can trigger proteolysis. Likewise, for correct polyubiquitylation of human RNAPII, NEDD4 cooperates with the ElonginA/B/C-Cullin 5 complex. These data indicate that RNAPII polyubiquitylation requires cooperation between distinct, sequentially acting ubiquitin ligases, and raise the intriguing possibility that other members of the large and functionally diverse family of NEDD4-like ubiquitin ligases also require the assistance of a second E3 when targeting proteins for degradation.elongin ͉ NEDD4 ͉ Rsp5 ͉ ubiquitylation P rotein ubiquitylation plays a crucial role in virtually all cell regulatory pathways. Mono-ubiquitylation commonly alters the activity of the target protein, or tags it for interaction with other factors, while the effect of polyubiquitylation depends on the type of ubiquitin chain being added. Ubiquitin lysine 48 (K48) chains most often result in degradation of the target protein by the proteasome, whereas other chains, such as those occurring through K63, are typically signals for proteolysisindependent pathways (1, 2).One interesting substrate for protein ubiquitylation is RNA-PII, which transcribes all protein-encoding genes in eukaryotes. Ubiquitylation and degradation of RNAPII was first thought to occur specifically in response to DNA damage (3-5), but more recent experiments have shown that RNAPII arrested during transcript elongation as a result of other transcription obstacles is also prone to ubiquitylation and degradation (6). Thus, degradation of RNAPII may be a ''last resort,'' used to clear active genes of persistently arrested RNAPII elongation complexes (6-9). Interestingly, the proteasome is nuclear and can be found on the coding region of genes by chromatin-immunoprecipitation (10), so RNAPII proteolysis may well occur on the DNA.We have reconstituted RNAPII ubiquitylation in vitro with highly purified, physiologically relevant yeast, or human, ubiquitylation factors, respectively (6,11,12). The yeast HECT E3 Rsp5 binds RNAPII via the flexible C-terminal repeat domain (CTD) of the Rpb1 subunit (13), but modifies the polymerase in the main body of the Rpb1 subunit (6,14). Mutation of RSP5 (rsp5-1; temperature-sensitiv...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation

Harreman

Täschner²,

Sigurdsson³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

152

143

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“…Rsp5 is the E3 ubiquitin ligase for DNA damage-induced ubiquitylation of RNAPII and binds the polymerase strongly, exclu-sively via the CTD (27,37,49). The physiological role, if any, of the Rsp5 stimulation of CTDK1 kinase activity remains to be determined.…”

Section: Dissociation Of Holo-rnapii Does Not Require Holo-tfiihmentioning

confidence: 99%

Hyperphosphorylation of the C-terminal Repeat Domain of RNA Polymerase II Facilitates Dissociation of Its Complex with Mediator

Max

Soegaard

Svejstrup

2007

Journal of Biological Chemistry

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107

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The Mediator complex associates with RNA polymerase II (RNAPII) at least partly via the RNAPII C-terminal repeat domain (CTD). This association greatly stimulates the CTD kinase activity of general transcription factor TFIIH, and subsequent CTD phosphorylation is involved in triggering promoter clearance. Here, highly purified proteins and a protein dissociation assay were used to investigate whether the RNAPII⅐Mediator complex (holo-RNAPII) can be disrupted by CTD phosphorylation, thereby severing one of the bonds that stabilize promoter-associated initiation complexes. We report that CTD phosphorylation by the serine 5-specific TFIIH complex, or its kinase module TFIIK, is indeed sufficient to dissociate holo-RNAPII. Surprisingly, phosphorylation by the CTD serine 2-specific kinase CTDK1 also results in dissociation. Moreover, the Mediator-induced stimulation of CTD phosphorylation previously reported for TFIIH is also observed with CTDK1 kinase. An unrelated CTD-binding protein, Rsp5, is capable of stimulating this CTD kinase activity as well. These data shed new light on mechanisms that drive the RNAPII transcription cycle and suggest a mechanism for the enhancement of CTD kinase activity by the Mediator complex. The C-terminal domain (CTD)2 of the largest RNA polymerase II subunit, Rpb1, is the target of dynamic phosphorylation/ dephosphorylation during the transcription cycle (1). A nonphosphorylated version of RNAPII enters promoters (2, 3), and the kinase activity associated with general transcription factor TFIIH somehow triggers promoter clearance by phosphorylation of serine 5 in the CTD repeat (Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 ) (4 -6). This is thought to be followed by phosphorylation of serine 2 by the CTDK1/pTEFb kinase and serine 5 dephosphorylation by Ssu72 phosphatase during transcript elongation (7-11). In all likelihood, Fcp1 phosphatase then removes the remaining CTD phosphorylation during or upon transcriptional termination, recycling the polymerase for a new round of transcription (12-15).The Mediator complex transduces signals from sequencespecific transcriptional regulators to the general transcription machinery (16 -18). The association of Mediator with RNAPII, and its function in transcription, depends on the RNAPII CTD (16). Mediator is able to bind an unphosphorylated glutathione S-transferase-CTD fusion protein in vitro (16) and can be displaced from RNAPII using the monoclonal antibody 8WG16, which specifically recognizes the CTD repeat (17,19). Apart from the CTD-interacting surfaces, Mediator also interacts with RNAPII domains outside of the CTD (20, 21).The observation that RNAPII enters the initiation complex in the unphosphorylated (RNAPIIA) form and leaves it in the hyperphosphorylated (RNAPIIO) form gave rise to the idea that the initiation to elongation transition is somehow facilitated by hyperphosphorylation of the CTD (3). The association with Mediator might be one of the mechanisms by which RNA-PIIA is tied to promoters. In crude yeast extracts, Mediator ...

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“…The E3 ligase recognizes the substrate protein and provides specificity to the reaction, although a recent study showed that an E2 may also function in target recognition (58). E3 ubiquitin ligases can be divided into two major classes that contain either a HECT (homologous to E6-AP carboxyl terminus) domain or a RING (really interesting new gene) finger domain and differ in the mechanism by which ubiquitin is transferred to the target protein.…”

Section: The Ubiquitin Machinerymentioning

confidence: 99%

G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

Marchese

Paing

Temple

et al. 2008

Annu. Rev. Pharmacol. Toxicol.

404

429

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The heptahelical G protein-coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine-and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.

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Communication between Distant Sites in RNA Polymerase II through Ubiquitylation Factors and the Polymerase CTD

Cited by 72 publications

References 28 publications

Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation

Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation

Hyperphosphorylation of the C-terminal Repeat Domain of RNA Polymerase II Facilitates Dissociation of Its Complex with Mediator

G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

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