Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.
As a key cellular regulatory protein p53 is subject to tight regulation by several E3 ligases. Here we demonstrate the role of HECT domain E3 ligase, WWP1, in regulating p53 localization and activity. WWP1 associates with p53 and induces p53 ubiquitination. Unlike other E3 ligases, WWP1 increases p53 stability; inhibition of WWP1 expression or expression of a ligase-mutant form results in decreased p53 expression. WWP1-mediated stabilization of p53 is associated with increased accumulation of p53 in cytoplasm with a concomitant decrease in its transcriptional activities. WWP1 effects are independent of Mdm2 as they are seen in cells lacking Mdm2 expression. Whereas WWP1 limits p53 activity, p53 reduces expression of WWP1, pointing to a possible feedback loop mechanism. Taken together, these findings identify the first instance of a ubiquitin ligase that causes stabilization of p53 while inactivating its transcriptional activities.
RNA polymerase II from the fission yeast Schizosaccharomyces pombe consists of 12 species of subunits, Rpb1–Rpb12. We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors. For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S-transferase (GST) sequence was fused to the rpb3 cDNA to produce GST–Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein. After successive affinity chromatography on glutathione and Ni2+ columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST–Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of the GST–Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex. Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3–Rpb8 interaction and consequently Rpb8 enhances Rpb1–Rpb3 interaction to some extent. We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit–subunit contacts.
RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.
The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY− mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.
Dietary Restriction (DR) extends longevity in diverse species suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses1. We have discovered a role for the HECT E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in C. elegans in response to diet restriction. We find that overexpression of wwp-1 in worms extends lifespan up to 20% under conditions of ad libitum feeding. This extension is dependent upon the FoxA transcription factor pha-4, and independent of the FoxO transcription factor, daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signaling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Additionally, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for DR induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate DR induced longevity.
The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY477 present in ezrin’s C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.
DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.
•Def1 protein is proteolytically processed in response to transcription stress•Processing is ubiquitin and proteasome dependent•Processed Def1 (pr-Def1) accumulates in the nucleus, where it binds RNAPII•Using its CUE domain, pr-Def1 then recruits Elongin-Cullin via Ela1’s UbH domain
DNA damage induces proteasome-dependent proteolytic processing of Def1, releasing an activated protein fragment, which highlights how irreversible proteasome-mediated cleavage of a protein can act as a posttranslational modification in this signaling pathway.
Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects
While the essential role of the adenosine deaminase ADAR2 in RNA editing is well established, how it is regulated remains largely unknown. Here, the prolyl isomerase Pin1 and the E3 ubiquitin ligase WWP2 are shown to play a role in regulating ADAR2 localisation and stability.
ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1−/− mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.
ADAR2; GluR2; Pin1; RNA editing; WWP2
The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.
Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.
HECT (homologous to the E6AP C terminus) ubiquitin ligases have diverse functions in eukaryotic cells. In screens for proteins that bind to the HECT ubiquitin ligase WWP1, we identified Spartin, which is also known as SPG20. This protein is truncated in a neurological disease, Troyer syndrome. In this study, we show that SPG20 associates with the surface of lipid droplets (LDs) and can regulate their size and number. SPG20 binds to another LD protein, TIP47, and both proteins compete with an additional LD protein, adipophilin/adipocyte differentiation-related protein, for occupancy of LDs. The mutant SPG20 present in Troyer syndrome does not possess these activities. Depletion of SPG20 using RNA interference increases the number and size of LDs when cells are fed with oleic acid. Binding of WWP1 to SPG20 and the consequent ubiquitin transfer remove SPG20 from LDs and reduce the levels of coexpressed SPG20. These experiments suggest functions for ubiquitin ligases and SPG20 in the regulation of LD turnover and potential pathological mechanisms in Troyer syndrome.
In Saccharomyces cerevisiae, RNA polymerase II assembly is probably initiated by the formation of the RPB3–RPB11 heterodimer. RPB3 is encoded by a single copy gene in the yeast, mouse and human genomes. The RPB11 gene is also unique in yeast and mouse, but in humans a gene family has been identified that potentially encodes several RPB11 proteins differing mainly in their C-terminal regions. We compared the abilities of both yeast and human proteins to heterodimerize. We show that the yeast RPB3/RPB11 heterodimer critically depends on the presence of the C-terminal region of RPB11. In contrast, the human heterodimer tolerates significant changes in RPB11 C-terminus, allowing two human RPB11 variants to heterodimerize with the same efficiency with RPB3. In keeping with this observation, the interactions between the conserved N-terminal ‘α-motifs’ is much more important for heterodimerization of the human subunits than for those in yeast. These data indicate that the heterodimerization interfaces have been modified during the course of evolution to allow a recent diversification of the human RPB11 subunits that remains compatible with heterodimerization with RPB3.
Treatment of Saccharomyces cerevisiae and human cells with DNA-damaging agents such as UV light or 4-nitroquinoline-1-oxide induces polyubiquitylation of the largest RNA polymerase II (Pol II) subunit, Rpb1, which results in rapid Pol II degradation by the proteasome. Here we identify a novel role for the yeast Elc1 protein in mediating Pol II polyubiquitylation and degradation in DNA-damaged yeast cells and propose the involvement of a ubiquitin ligase, of which Elc1 is a component, in this process. In addition, we present genetic evidence for a possible involvement of Elc1 in Rad7-Rad16-dependent nucleotide excision repair (NER) of lesions from the nontranscribed regions of the genome and suggest a role for Elc1 in increasing the proficiency of repair of nontranscribed DNA, where as a component of the Rad7-Rad16-Elc1 ubiquitin ligase, it would promote the efficient turnover of the NER ensemble from the lesion site in a Rad23-19S proteasomal complex-dependent reaction.
Treatment of yeast and human cells with DNA-damaging agents elicits lysine 48-linked polyubiquitylation of Rpb1, the largest subunit of RNA polymerase II (Pol II), which targets Pol II for proteasomal degradation. However, the ubiquitin ligase (E3) responsible for Pol II polyubiquitylation has not been identified in humans or the yeast Saccharomyces cerevisiae . Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in yeast cells, and on the basis of these and other observations, we propose that an E3 comprised of elongin C (Elc1), Ela1, Cul3, and the RING finger protein Roc1 (Rbx1) mediates this process in yeast cells. This study provides, in addition to the identification of the E3 required for Pol II polyubiquitylation and degradation in yeast cells, the first evidence for a specific function in yeast for a member of the elongin C/BC-box protein/cullin family of ligases. Also, these observations raise the distinct possibility that the elongin C-containing ubiquitin ligase, the von Hippel-Lindau tumor suppressor complex, promotes Pol II polyubiquitylation and degradation in human cells.
A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.
Eukaryotic nuclei contain three classes of multisubunit DNA-directed RNA polymerase. At the core of each complex is a set of 12 highly conserved subunits of which five—RPB5, RPB6, RPB8, RPB10, and RPB12—are thought to be common to all three polymerase classes. Here, we show that four distantly related eukaryotic lineages (the higher plant and three protistan) have independently expanded their repertoire of RPB5 and RPB6 subunits. Using the protozoan parasite Trypanosoma brucei as a model organism, we demonstrate that these distinct RPB5 and RPB6 subunits localize to discrete subnuclear compartments and form part of different polymerase complexes. We further show that RNA interference-mediated depletion of these discrete subunits abolishes class-specific transcription and hence demonstrates complex specialization and diversification of function by conventionally shared subunit groups.
Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.
Late domains are short peptide sequences encoded by enveloped viruses to promote the final separation of the nascent virus from the infected cell. These amino acid motifs facilitate viral egress by interacting with components of the ESCRT (endosomal sorting complex required for transport) machinery, ultimately leading to membrane scission by recruiting ESCRT-III to the site of viral budding. PPXY late (L) domains present in viruses such as murine leukemia virus (MLV) or human T-cell leukemia virus type 1 (HTLV-1) access the ESCRT pathway via interaction with HECT ubiquitin ligases (WWP1, WWP2, and Itch). However, the mechanism of ESCRT-III recruitment in this context remains elusive. In this study, we tested the arrestin-related trafficking (ART) proteins, namely, ARRDC1 (arrestin domain-containing protein 1) to ARRDC4 and TXNIP (thioredoxin-interacting protein), for their ability to function as adaptors between HECT ubiquitin ligases and the core ESCRT machinery in PPXY-dependent budding. We present several lines of evidence in support of such a role: ARTs interact with HECT ubiquitin ligases, and they also exhibit multiple interactions with components of the ESCRT pathway, namely, ALIX and Tsg101, and perhaps with an as yet unidentified factor. Additionally, the ARTs can be recruited to the site of viral budding, and their overexpression results in a PPXY-specific inhibition of MLV budding. Lastly, we show that WWP1 changes the ubiquitination status of ARRDC1, suggesting that the ARTs may provide a platform for ubiquitination in PPXY-dependent budding. Taken together, our results support a model whereby ARTs are involved in PPXY-mediated budding by interacting with HECT ubiquitin ligases and providing several alternative routes for ESCRT-III recruitment.
To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.
Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-β25/26 (prolonging the Bridge helix) and Rpb1-α44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Δ or its RNA polymerase I rpa12Δ counterpart. Rpb1-β25/26, Rpb1-α44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-α46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5–Rpb1 binding and the Rpb9–Rpb1 interaction is to hold the Bridge helix, the Rpb1-α44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.
Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions.
Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.
Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.
Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.
We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.