Unconventional Prefoldin RPB5 Interactor Research Articles

The prefoldin-like protein AtURI exhibits characteristics of intrinsically disordered proteins.

The prefoldin-like protein UNCONVENTIONAL PREFOLDIN RPB5 INTERACTOR (URI) participates in diverse cellular functions, including protein homeostasis, transcription, translation, and signal transduction. Thus, URI is a highly versatile protein, although the molecular basis of this versatility remains unknown. In this work, we show that Arabidopsis thaliana (Arabidopsis) URI (AtURI) possesses a large intrinsically disordered region (IDR) spanning most of the C-terminal part of the protein, a feature conserved in yeast and human orthologs. Our findings reveal two key characteristics of disordered proteins in AtURI: promiscuity in interacting with partners and protein instability. We propose that these two features contribute to providing AtURI with functional versatility.

URI alleviates tyrosine kinase inhibitors-induced ferroptosis by reprogramming lipid metabolism in p53 wild-type liver cancers

The clinical benefit of tyrosine kinase inhibitors (TKIs)-based systemic therapy for advanced hepatocellular carcinoma (HCC) is limited due to drug resistance. Here, we uncover that lipid metabolism reprogramming mediated by unconventional prefoldin RPB5 interactor (URI) endows HCC with resistance to TKIs-induced ferroptosis. Mechanistically, URI directly interacts with TRIM28 and promotes p53 ubiquitination and degradation in a TRIM28-MDM2 dependent manner. Importantly, p53 binds to the promoter of stearoyl-CoA desaturase 1 (SCD1) and represses its transcription. High expression of URI is correlated with high level of SCD1 and their synergetic expression predicts poor prognosis and TKIs resistance in HCC. The combination of SCD1 inhibitor aramchol and deuterated sorafenib derivative donafenib displays promising anti-tumor effects in p53-wild type HCC patient-derived organoids and xenografted tumors. This combination therapy has potential clinical benefits for the patients with advanced HCC who have wild-type p53 and high levels of URI/SCD1.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two β-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation.

Intestinal epithelium regenerates rapidly through proliferation of intestinal stem cells (ISCs), orchestrated by potent mitogens secreted within the crypt niche. However, mechanisms regulating these mitogenic factors remain largely unknown. Here, we demonstrate that transit-amplifying (TA) cells, marked by unconventional prefoldin RPB5 interactor (URI), control R-spondin production to guide ISC proliferation. Genetic intestinal URI ablation in mice injures TA cells, reducing their survival capacity, leading to an inflamed tissue and subsequently decreasing R-spondin levels, thereby causing ISC quiescence and disruption of intestinal structure. R-spondin supplementation or restoration of R-spondin levels via cell death inhibition by c-MYC elimination or the suppression of inflammation reinstates ISC proliferation in URI-depleted mice. However, selective c-MYC and p53 suppression are required to fully restore TA cell survival and differentiation capacity and preserve complete intestinal architecture. Our data reveal an unexpected role of TA cells, which represent a signaling platform instrumental for controlling inflammatory cues and R-spondin production, essential for maintaining ISC proliferation and tissue regeneration.

CircURI1 interacts with hnRNPM to inhibit metastasis by modulating alternative splicing in gastric cancer

Circular RNAs (circRNAs) have emerged as key regulators of human cancers, yet their modes of action in gastric cancer (GC) remain largely unknown. Here, we identified circURI1 back-spliced from exons 3 and 4 of unconventional prefoldin RPB5 interactor 1 (URI1) from circRNA profiling of five-paired human gastric and the corresponding nontumor adjacent specimens (paraGC). CircURI1 exhibits the significantly higher expression in GC compared with paraGC and inhibitory effects on cell migration and invasion in vitro and GC metastasis in vivo. Mechanistically, circURI1 directly interacts with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to modulate alternative splicing of genes, involved in the process of cell migration, thus suppressing GC metastasis. Collectively, our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.

URI1 suppresses irradiation-induced reactive oxygen species (ROS) by activating autophagy in hepatocellular carcinoma cells.

Radiotherapy has been extensively applied in cancer treatment. However, this treatment is ineffective in Hepatocellular carcinoma (HCC) due to lack of radiosensitivity. Unconventional prefoldin RPB5 interactor 1 (URI1) exhibits characteristics similar to those oncoproteins, which promotes survival of cancer cells. As a consequence of the irradiation, the levels of endogenous reactive oxygen species (ROS) rise. In the current study, we analyzed the role of URI1 in the control of ROS levels in HepG2 cells. Upon URI1 overexpression, HepG2 cells significantly suppressed irradiation-induced ROS, which may help cells escape from oxidative toxicity. And our data demonstrated that overexpression of URI1 not only resulted in an increase of autophagic flux, but also resulted in an further increased capacity of autophagy to eliminate ROS. It indicated that URI1 suppressed irradiation-induced ROS through activating autophagy. Moreover, URI1 activated autophagy by promoting the activities of AMP-activated protein kinase (AMPK). Results showed that overexpression of URI1 increased the phosphorylation of AMPKα at the Thr172 residue and the activated-AMPK promoted the phosphorylation of forkhead box O3 (FOXO3) at the Ser253 residue, which significantly induced autophagy. Taken together, our findings provide a mechanism that URI1 suppresses irradiation-induced ROS by activating autophagy through AMPK/FOXO3 signaling pathway. These new molecular insights will provide an important contribution to our better understanding about irradiation insensitivity of HCC.

Coxsackievirus B Type 4 Infection in β Cells Downregulates the Chaperone Prefoldin URI to Induce a MODY4-like Diabetes via Pdx1 Silencing

SummaryEnteroviruses are suspected to contribute to insulin-producing β cell loss and hyperglycemia-induced diabetes. However, mechanisms are not fully defined. Here, we show that coxsackievirus B type 4 (CVB4) infection in human islet-engrafted mice and in rat insulinoma cells displays loss of unconventional prefoldin RPB5 interactor (URI) and PDX1, affecting β cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in β cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in β cells are more glucose tolerant. Mechanistically, URI loss triggers estrogen receptor nuclear translocation leading to DNA methyltransferase 1 (DNMT1) expression, which induces Pdx1 promoter hypermethylation and silencing. Consequently, demethylating agent procainamide-mediated DNMT1 inhibition reinstates PDX1 expression and protects against diabetes in pancreatic URI-depleted mice . Finally, the β cells of human diabetes patients show correlations between viral protein 1 and URI, PDX1, and DNMT1 levels. URI and DNMT1 expression and PDX1 silencing provide a causal link between enterovirus infection and diabetes.

URI-1 Levels Are Elevated in Fatty Livers of db/db and C57BL/6 Mice Fed Trans-10, Cis-12 Conjugated Linoleic Acid

Abstract Objectives This study was designed to investigate whether unconventional prefoldin RPB5 interactor (URI)-1 mediates hepatic accumulation of triglyceride (TG) in response to a diet with trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) in lean or genetically obese mice. URI-1 belongs to the prefoldin family of proteins that have been shown to coordinate nutrient availablility by transcriptional regulation of genes involved in glucose and lipid metabolism. Thus, it was hypothesized that URI-1 in liver is involved in increased fatty acid uptake and accumulation leading to fatty liver. Methods C57BL/6 and db/db mice were randomly assigned to two diet groups, control (CTL) and t10,c12 CLA (0.4% w/w). After 4 weeks, the mice were weighed and euthanized. Livers were dissected, weighed and stored at –80°C. Liver lysates were prepared from the tissue for Western blotting to measure hepatic protein levels of URI-1 and FABP1. The amount of lipid in the livers was determined using the LabAssay™ Triglyceride kit, a colorimetric TG assay. Results The liver to body weight ratio of db/db and C57BL/6 mice fed t10,c12 CLA increased by 90% and 52%, respectively, compared to their counterparts fed the CTL diet. Likewise, the hepatic TG concentration (mg TG/mg protein) was increased 38% and 5-fold, respectively, in CLA-fed db/db and C57BL/6 mice compared to CTL db/db and C57BL/6 mice. Western blotting showed that FABP1 levels were approximately 2-fold greater in the db/db t10,c12 CLA group relative to the db/db CTL group, and may contribute to increased fatty acid uptake. Furthermore, URI-1 protein levels were elevated 4-fold in db/db and C57BL6 mice fed t10,c12 CLA compared to their respective CTL groups. Lastly, correlation analysis revealed that URI-1 levels were significantly correlated with hepatic TG concentrations (r = 0.61) and liver/body weight ratio (r = 0.64). Conclusions This study revealed a relationship between hepatic TG accumulation and URI-1, a protein associated with hepatocellular carcinoma (HCC) and cirrhosis. This study provides a basis for in vitro experiments exploring the causative role of URI-1 in propagating hepatic TG accumulation, and ultimately the progression of fatty liver disease to HCC and cirrhosis. Funding Sources University Collaborative Research Project, NSERC Discovery, and University of Manitoba Graduate Enhancement of Tri-Council Stipends.

URI is required to maintain intestinal architecture during ionizing radiation.

Ionizing radiation (IR) can cause gastrointestinal syndrome (GIS), a lethal disorder, by means of unknown mechanisms. We show that high-dose irradiation increases unconventional prefoldin RPB5 interactor (URI) levels in mouse intestinal crypt, but organ regeneration correlates with URI reductions. URI overexpression in intestine protects mice from radiation-induced GIS, whereas halving URI expression sensitizes mice to IR. URI specifically inhibits β-catenin in stem cell-like label-retaining (LR) cells, which are essential for organ regeneration after IR. URI reduction activates β-catenin-induced c-MYC expression, causing proliferation of and DNA damage to LR cells, rendering them radiosensitive. Therefore, URI labels LR cells which promote tissue regeneration in response to high-dose irradiation, and c-MYC inhibitors could be countermeasures for humans at risk of developing GIS.

BRAF inhibition sensitizes melanoma cells to \u03b1-amanitin via decreased RNA polymerase II assembly

Despite the great success of small molecule inhibitors in the treatment of patients with BRAFV600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAFV600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAFV600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAFV600E inhibitors.

The Expression of MicroRNA-598 Inhibits Ovarian Cancer Cell Proliferation and Metastasis by Targeting URI

Unconventional prefoldin RPB5 interactor (URI, or RMP, a member of the prefoldin family of molecular chaperones) exhibits oncogenic activity in several types of cancer, including ovarian cancer. However, the underlying regulatory mechanism in ovarian cancer remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To elucidate the role of miRNAs in URI-induced ovarian cancer, miR-598 and URI were overexpressed in the SKOV3 ovarian cancer cell line. The CCK8 kit was used to determine cell proliferation, and the Transwell assay was used to measure cell invasion and migration. RT-PCR and western blotting were used to analyze the expression of miR-598 and URI, and the luciferase reporter assay was used to examine the interaction between miR-598 and URI. Nude mice were used to characterize the regulation of tumor growth in vivo. The results showed that the expression of miR-598 inhibited the proliferation, invasion, and migration of ovarian cancer cells by targeting URI. The inhibitory effect of miR-598 was reversed by overexpression of URI. The luciferase reporter assay showed that miR-598 downregulated URI by directly targeting the 3′ UTR of URI. In vivo studies showed that the expression of miR-598 significantly inhibited the growth of tumors. Taken together, the results suggested that miR-598 inhibited tumor growth and metastasis by targeting URI.

The Yeast Prefoldin Bud27.

Bud27 and its human orthologue URI (unconventional prefoldin RPB5-interactor) are members of the prefoldin (PFD) family of ATP-independent molecular chaperones binding the Rpb5 subunit to all three nuclear eukaryotic RNA polymerases (RNA pols). Bud27/URI are considered to function as a scaffold protein able to assemble additional members of the prefoldin (PDF) family in both human and yeast. Bud27 and URI are not subunits of the canonical PFD/GimC complex and not only the composition but also other functions independent of the PFD/GimC complex have been described for Bud27 and URI. Bud27 interacts only with Pfd6 but no other components of the R2TP/PFDL. Furthermore previously reported interaction between Bud27 and Pfd2 was not later confirmed. These results point to major differences in the prefoldin-like complex composition between yeast and other organisms, suggesting also important differences in functions. Furthermore, this assumption could be extended to the R2TP/PFDL complex, which has been shown to differ between different organisms and has not been identified in yeast. This casts doubt on whether Bud27 cooperation with prefoldin and other components of the R2TP/PFDL modules are required for its action. This could be extended to URI and point to a role of Bud27/URI in cell functions more relevant than this previously proposed as co-prefoldin.

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

Molecular cross-talk between the liver and white adipose tissue links excessive noURIshment to hepatocellular carcinoma

In their recent study published in Cancer Cell , Gomes and colleagues uncovered a novel mechanism driving obesity-triggered hepatocellular carcinoma (HCC). The authors revealed a feed-forward loop between hepatic unconventional prefoldin RPB5 interactor (URI) and cytokine interleukin-17A (IL-17A), and showed that chronically high expression of both proteins induces DNA damage, followed by systemic inflammation, which initiates non-alcoholic steatohepatitis (NASH) and HCC. URI and IL-17A are involved in cross-talk between the liver and white adipose tissue (WAT), with lipolysis, neutrophil infiltration and insulin resistance occurring in WAT, resulting in hepatosteatosis, injury and HCC in the liver. These results suggest that targeting URI and IL-17 levels may be a useful therapeutic strategy in the treatment of obesity-driven HCC.

Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma

Obesity increases hepatocellular carcinoma (HCC) risks via unknown mediators. We report that hepatic unconventional prefoldin RPB5 interactor (URI) couples nutrient surpluses to inflammation and non-alcoholic steatohepatitis (NASH), a common cause of HCC. URI-induced DNA damage in hepatocytes triggers inflammation via T helper 17 (Th17) lymphocytes and interleukin 17A (IL-17A). This induces white adipose tissue neutrophil infiltration mediating insulin resistance (IR) and fatty acid release, stored in liver as triglycerides, causing NASH. NASH and subsequently HCC are prevented by pharmacological suppression of Th17 cell differentiation, IL-17A blocking antibodies, and genetic ablation of the IL-17A receptor in myeloid cells. Human hepatitis, fatty liver, and viral hepatitis-associated HCC exhibit increased IL-17A correlating positively with steatosis. IL-17A blockers may prevent IR, NASH, and HCC in high-risk patients.

Colorectal cancer cells display chaperone dependency for the unconventional prefoldin URI1.

Chaperone dependency of cancer cells is an emerging trait that relates to the need of transformed cells to cope with the various stresses associated with the malignant state. URI1 (unconventional prefoldin RPB5 interactor 1) encodes a member of the prefoldin (PFD) family of molecular chaperones that acts as part of a heterohexameric PFD complex, the URI1 complex (URI1C), to promote assembly of multiprotein complexes involved in cell signaling and transcription processes. Here, we report that human colorectal cancer (CRCs) cell lines demonstrate differential dependency on URI1 and on the URI1 partner PFD STAP1 for survival, suggesting that this differential vulnerability of CRC cells is directly linked to URI1C chaperone function. Interestingly, in URI1-dependent CRC cells, URI1 deficiency is associated with non-genotoxic p53 activation and p53-dependent apoptosis. URI1-independent CRC cells do not exhibit such effects even in the context of wildtype p53. Lastly, in tumor xenografts, the conditional depletion of URI1 in URI1-dependent CRC cells was, after tumor establishment, associated with severe inhibition of subsequent tumor growth and activation of p53 target genes. Thus, a subset of CRC cells has acquired a dependency on the URI1 chaperone system for survival, providing an example of ‘non-oncogene addiction’ and vulnerability for therapeutic targeting.

Oncogene-induced NAD(+) depletion in tumorigenesis.

Oncogene-induced NAD(+) depletion in tumorigenesis.

NAD+ Supplementation as a Novel Approach to cURIng HCC?

In this issue of Cancer Cell, Tummala and colleagues demonstrate that unconventional prefoldin RPB5 interactor (URI) expression in hepatocytes leads to hepatocellular carcinoma (HCC) development by interacting with L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism. The results suggest that supplementation of NAD(+) may be a prophylactic or therapeutic approach in HCC.

Inhibition of De Novo NAD+ Synthesis by Oncogenic URI Causes Liver Tumorigenesis through DNA Damage

Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC.

Biological function and molecular mechanism of URI in HepG2 cells

To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells. The cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors. Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line. The cell proliferation and anchor-independent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay. Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells. Apoptosis related genes were detected by Western blot. The pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained. Cell proliferation rates of the HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were (588.78 ± 32.12)%, (959.33 ± 58.8)% and (393.93 ± 39.7)%, respectively (P < 0.05). The number of cell clones of HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7, 85 ± 5 and 20 ± 4 (P < 0.05), respectively. After γ-ray irradiation, the URI-overexpressing cell line showed a significantly lower apoptosis rate and G(2)/M phase arrest than those in the URI-depleted cell line (P < 0.05). In the HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.92 ± 0.03, 1.11 ± 0.13 and 0.82 ± 0.01 (P < 0.05). In the pCDNA3.1-URI-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 1.79 ± 0.12, 0.48 ± 0.01 and 2.20 ± 0.30 (P < 0.05), respectively. In the pGPU6-URIi-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.50 ± 0.04, 1.52 ± 0.20 and 0.38 ± 0.01 (P < 0.05), respectively. The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line. However, on the contrary, expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line. URI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro. After the impairment of URI expression, the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist γ-ray irradiation is reduced. URI may become a potential new target for cancer therapy of hepatocellular carcinoma.