RACK1 and IRE1 participate in the translational quality control of amyloid precursor protein in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identidying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. In this study, we show that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. We searched for regulators that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. Our results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD.

RACK1 promotes the occurrence and progression of cervical carcinoma.

BACKGROUND: RACK1 has been identified as a multifunctional cytosolic protein, and plays a pivotal role in multiple biological responses involved in several kinds of tumors, while its effect in cervical cancer has not been well elucidated yet. The study aimed to investigate the role of RACK1 in cervical cancer occurrence and progression. METHODS: The expression of RACK1 in cervical specimens was measured by immunohistochemical staining and Western blot assay. Transgenic mice were used to detect the role of RACK1 in modulating tumorigenesis in vivo. Cervical carcinoma cell lines were used to explore the underlying mechanisms of RACK1 on the behaviors of tumor cells in vitro. RESULTS: We found that RACK1 expression was upregulated in cancer tissues compared with adjacent tissues, and its expression was gradually increased from cervictis, and cervical intraepithelial neoplasis (CIN) to carcinoma. Genetic overexpression of RACK1 facilitated tumor formation and growth in nude mice. Mechanism studies disclosed that RACK1 over-expression prolonged the G0 /G1 phase by up-regulating the expression of cyclinD1, down-regulating p21 and p27 probably by modulating the phosphorylation of AKT. CONCLUSIONS: Taken together, we concluded that RACK1 stimulates tumorigenesis and progression of cervical cancer via modulating the proliferation of tumor cells, implying that targeting RACK1 may serve as a promising method for cervical cancer therapy.

Toxoplasma gondii surface antigen 1 (SAG1) interacts in vitro with host cell receptor for activated C kinase 1 (RACK1).

Toxoplasma gondii (T. gondii) surface antigen 1 (SAG1) is crucial for tachyzoite invasion into host cells. However, the role of SAG1 in interaction with host cells remains unknown. The primary objective of this study was to analyze and validate the interaction between SAG1 and host cells. RACK1, an intracellular multifunctional protein, was identified as a SAG1 binding partner in host cells. Furthermore, the expression of RACK1 is manipulated by SAG1, and depletion of RACK1 negatively regulated host cell viability. These results imply that through interaction with RACK1, SAG1 preserves the viability of host cells to satisfy the survival needs of T. gondii. Our findings suggest a novel role for SAG1 in intracellular parasitism.

Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

TAR DNA-binding protein 43 (TDP-43) and Fused in Sarcoma/Translocated in Sarcoma (FUS) are ribonucleoproteins associated with pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under physiological conditions, TDP-43 and FUS are predominantly localized in the nucleus, where they participate in transcriptional regulation, RNA splicing and metabolism. In disease, however, they are typically mislocalized to the cytoplasm where they form aggregated inclusions. A number of shared cellular pathways have been identified that contribute to TDP-43 and FUS toxicity in neurodegeneration. In the present study, we report a novel pathogenic mechanism shared by these two proteins. We found that pathological FUS co-aggregates with a ribosomal protein, the Receptor for Activated C-Kinase 1 (RACK1), in the cytoplasm of spinal cord motor neurons of ALS, as previously reported for pathological TDP-43. In HEK293T cells transiently transfected with TDP-43 or FUS mutant lacking a functional nuclear localization signal (NLS; TDP-43ΔNLS and FUSΔNLS), cytoplasmic TDP-43 and FUS induced co-aggregation with endogenous RACK1. These co-aggregates sequestered the translational machinery through interaction with the polyribosome, accompanied by a significant reduction of global protein translation. RACK1 knockdown decreased cytoplasmic aggregation of TDP-43ΔNLS or FUSΔNLS and alleviated associated global translational suppression. Surprisingly, RACK1 knockdown also led to partial nuclear localization of TDP-43ΔNLS and FUSΔNLS in some transfected cells, despite the absence of NLS. In vivo, RACK1 knockdown alleviated retinal neuronal degeneration in transgenic Drosophila melanogaster expressing hTDP-43WT or hTDP-43Q331K and improved motor function of hTDP-43WT flies, with no observed adverse effects on neuronal health in control knockdown flies. In conclusion, our results revealed a novel shared mechanism of pathogenesis for misfolded aggregates of TDP-43 and FUS mediated by interference with protein translation in a RACK1-dependent manner. We provide proof-of-concept evidence for targeting RACK1 as a potential therapeutic approach for TDP-43 or FUS proteinopathy associated with ALS and FTLD.

Nuclear Localization of G3BP6 Is Essential for the Flowering Transition in Arabidopsis.

The Ras GTPase-activating protein SH3 domain-binding protein (G3BP) belongs to the highly conserved family of RNA-binding proteins, which has been well-investigated in humans and animals. However, limited study of plant G3BP has been reported, and the precise biological function of the G3BP family has not been elucidated yet. In this study, the Arabidopsis G3BP family, comprising seven members, was comparatively analyzed. Transcriptome analysis showed that most G3BP genes are ubiquitously expressed in various tissues/organs. Transient expression analysis revealed that all G3BPs were presented in the cytoplasm, among which G3BP6 was additionally found in the nucleus. Further study revealed a conserved NLS motif required for the nuclear localization of G3BP6. Additionally, phenotypic analysis revealed that loss-of-function g3bp6 presented late-flowering phenotypes. RNA-sequencing analysis and qRT-PCR assays demonstrated that the expressions of abundant floral genes were significantly altered in g3bp6 plants. We also discovered that overexpression of G3BP6 in the nucleus, rather than in the cytoplasm, propelled bolting. Furthermore, we revealed that the scaffold protein Receptor for Activated C Kinase 1 (RACK1) interacted with and modulated the nuclear localization of G3BP6. Altogether, this study sheds new light on G3BP6 and its specific role in regulating the flowering transition in Arabidopsis.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Loss of SMURF2 expression enhances RACK1 stability and promotes ovarian cancer progression.

Receptor for activated C kinase 1 (RACK1) has been confirmed to take part in multiple biological events and the mechanism supporting abnormal RACK1 expression in ovarian cancer (OC) remains to be characterized. Here, we identified Smad ubiquitin regulatory factor 2 (SMURF2) as a bona fide E3 ligase of RACK1 in OC. SMURF2 effectively added the K6, K33 and K48 ubiquitin chains to the RACK1, resulting in polyubiquitination and instability of RACK1. PCAF promoted acetylation of RACK1 at K130, leading to SMURF2-mediated RACK1 ubiquitination inhibited and promote OC progression. The expression levels of SMURF2 and RACK1 were negatively correlated. SMURF2 was abnormal low expression in human ovarian cancer, resulting in decreased ubiquitination of RACK1 and increased stability, which promoted OC progression, and strongly associated with poor patients' prognosis. In general, our results demonstrated that SMURF2 plays a pivotal role in stabilizing RACK1, which in turn facilitates tumorigenesis in OC, suggesting that SMURF2-RACK1 axis may prove to be potential targets for the treatment of OC.

Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila.

Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, we reveal that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, our present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity.

Neuropilin 1 promotes unilateral ureteral obstruction-induced renal fibrosis via RACK1 in renal tubular epithelial cells.

Neuropilin 1 (NRP1) is a single-channel transmembrane glycoprotein whose role and mechanism in renal fibrosis remain incompletely elucidated. Therefore, we investigated the effect of NRP1 on renal fibrosis and its potential mechanism. NRP1 expression in the renal sections from patients with chronic kidney disease (CKD) and a unilateral ureteral obstruction (UUO) mouse model was detected. Nrp1 overexpression or knockdown plasmid was transfected into mice, TKPTS mouse kidney proximal tubular epithelial cells (TECs), and rat kidney fibroblasts, after which pathological injury evaluation and fibrosis marker detection were conducted. The direct interaction of the receptor of activated protein C kinase 1 (RACK1) with NRP1 was validated by immunoprecipitation and Western blot analysis. We found that the upregulated renal NRP1 expression in patients with CKD was located in proximal TECs, consistent with the degree of interstitial fibrosis. In the UUO mouse model, NRP1 expression was upregulated in the kidney, and overexpression of Nrp1 increased the mRNA and protein expression of fibronectin (Fn) and α-smooth muscle actin (α-SMA), whereas Nrp1 knockdown significantly reduced Fn and α-SMA expression and downregulated the inflammatory response. NRP1 promoted transforming growth factor ß1 (TGF-ß1)-induced profibrotic responses in the TKPTS cells and fibroblasts, and Nrp1 knockdown partially reversed these responses. Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry verified that NRP1 can directly bind to RACK1, and Rack1 knockdown reversed the NRP1-induced fibrotic response. In summary, NRP1 may enhance the TGF-ß1 pathway by binding to RACK1, thus promoting renal fibrosis.NEW & NOTEWORTHY Although a few studies have confirmed the correlation between neuropilin 1 (NRP1) and renal diseases, the mechanism of NRP1 in renal fibrosis remains unclear. Here, we investigated the effects of NRP1 on renal fibrosis through in vitro and in vivo experiments and explored the possible downstream mechanisms. We found that NRP1 can stimulate the TGF-ß1 signaling pathway, possibly by binding to RACK1, thereby promoting renal fibrosis.

Research of STEAP3 interaction with Rab7A and RACK1 to modulate the MAPK and JAK/STAT signaling in Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. The molecular mechanisms underlying OA progression remain incompletely understood. In this study, we investigated the role of STEAP3 (Six Transmembrane Epithelial Antigen of the Prostate 3) in the development of OA. Our results demonstrated that STEAP3 was upregulated in OA cartilage tissues and contributes to the progression of the disease. To elucidate the mechanism, we employed transcriptomic and interaction proteomics analysis, and identified dysregulated genes and pathways associated with STEAP3 overexpression. Specifically, we found that STEAP3 interacted with Rab7A, a protein involved in intracellular trafficking and autophagy, and suppressed its activity. In addition, STEAP3 interacted with activated C kinase 1 (RACK1) and enhanced its activity. Furthermore, our data indicated that the suppression of Rab7A activity by STEAP3 promoted the activation of receptor tyrosine kinases (RTKs) and the promoting effects of RACK1 by STEAP3, both of which in turn activated the MAPK and JAK/STAT signaling pathways. In conclusion, our findings highlighted the role of STEAP3 in promoting OA progression. By inhibiting Rab7A activity and promoting RACK1 activity, STEAP3 enhanced inflammation through the activation of RTKs and subsequent activation of the MAPK and JAK/STAT signaling pathways. Targeting STEAP3 may provide a potential therapeutic strategy for the treatment of OA by modulating these interconnected pathways.

Prognostic and clinicopathological role of RACK1 for cancer patients: a systematic review and meta-analysis.

Background: The receptor for activated C kinase 1 (RACK1) expression is associated with clinicopathological characteristics and the prognosis of various cancers; however, the conclusions are controversial. As a result, this study aimed to explore the clinicopathological and prognostic values of RACK1 expression in patients with cancer. Methodology: PubMed, Embase, Web of Science, Cochrane Library, and Scopus were comprehensively explored from their inception to April 20, 2023, for selecting studies on the clinicopathological and prognostic role of RACK1 in patients with cancer that met the criteria for inclusion in this review. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were used to assess the prognosis-predictive value of RACK1 expression, while pooled odds ratios (ORs) and 95% CIs were used to evaluate the correlation between RACK1 expression and the clinicopathological characteristics of patients with cancer. The quality of the included studies was evaluated using the Newcastle-Ottawa Scale. Results: Twenty-two studies (13 on prognosis and 20 on clinicopathological characteristics) were included in this systematic review and meta-analysis. The findings indicated that high RACK1 expression was significantly associated with poor overall survival (HR = 1.62; 95% CI, 1.13-2.33; P = 0.009; I2 = 89%) and reversely correlated with disease-free survival/recurrence-free survival (HR = 1.87; 95% CI, 1.22-2.88; P = 0.004; I2 = 0%). Furthermore, increased RACK1 expression was significantly associated with lymphatic invasion/N+ stage (OR = 1.74; 95% CI, 1.04-2.90; P = 0.04; I2 = 79%) of tumors. Conclusions: RACK1 may be a global predictive marker of poor prognosis in patients with cancer and unfavorable clinicopathological characteristics. However, further clinical studies are required to validate these findings.

TRIM26 inhibited osteosarcoma progression through destabilizing RACK1 and thus inactivation of MEK/ERK signaling.

Osteosarcoma is a highly aggressive malignant tumor that is common in the pediatric population and has a high rate of disability and mortality. Recent studies have suggested that the tripartite motif-containing family genes (TRIMs) play critical roles in oncogenesis in several cancers. TRIM26, one of the TRIMs family genes, was more frequently reported to exert a tumor-suppressive role, while its detailed functional roles in the osteosarcoma progression were still unknown and require further investigation. Herein, we found that TRIM26 was markedly downregulated in osteosarcoma tissues and cells. Survival analysis revealed that higher expression of TRIM26 was associated with better prognosis and its expression was an independent protective factor in osteosarcoma. Functional analysis demonstrated that overexpression of TRIM26 inhibited osteosarcoma cell proliferation and invasion via inhibiting the EMT process and MEK/ERK signaling. In contrast, the silence of TRIM26 caused the opposite effect. RACK1, a member of the Trp-Asp repeat protein family, was identified as a novel target of TRIM26. TRIM26 could interact with RACK1 and accelerate the degradation of RACK1, thus inactivation of MEK/ERK signaling. Overexpression of RACK1 could attenuate the inhibitory effect of TRIM26 overexpression on p-MEK1/2 and p-ERK1/2, and silence of RACK1 could partly impair the effect of TRIM26 knockdown-induced upregulation of p-MEK1/2 and p-ERK1/2. Further, a series of gain- and loss-of-function experiments showed that decreased malignant behaviors including cell proliferation and invasion in TRIM26-upregulated cells were reversed when RACK1 was overexpressed, whereas RACK1 knockdown diminished the increased malignant phenotypes in TRIM26-silenced osteosarcoma cells. In conclusion, our study indicated that TRIM26 inhibited osteosarcoma progression via promoting proteasomal degradation of RACK1, thereby resulting in inactivation of MEK/ERK signaling, and impeding the EMT process.

OTUB1's role in promoting OSCC development by stabilizing RACK1 involves cell proliferation, migration, invasion, and tumor-associated macrophage M1 polarization.

Ovarian tumor domain, ubiquitin aldehyde binding 1 (OTUB1), a deubiquitinating enzyme known to regulate the stability of downstream proteins, has been reported to regulate various cancers tumorigenesis, yet its direct effects on oral squamous cell carcinoma (OSCC) progression are unclear. Bioinformatics analysis was performed to screen for genes of interest, and in vitro and in vivo studies were carried out to investigate the function and mechanism of OTUB1 in OSCC. We found that OTUB1 was abnormally elevated in OSCC tissues and positively associated with the pathological stage and tumor stage. Knockdown of OTUB1 impaired the malignance of OSCC cells - suppressed cell proliferation, invasion, migration, and xenografted tumor growth. OTUB1 silencing also drove tumor-associated macrophage M1 polarization but suppressed M2 polarization, and the induction of M1 polarization inhibited the survival of OSCC cells. However, OTUB1 overexpression exerted the opposite effects. Furthermore, the protein network that interacted with the OTUB1 protein was constructed based on the GeneMANIA website. Receptor for activated C kinase 1 (RACK1), a facilitator of OSCC progression, was identified as a potential target of the OTUB1 protein. We revealed that OTUB1 positively regulated RACK1 expression and inhibited RACK1 ubiquitination. Additionally, RACK1 upregulation reversed the effects of OTUB1 knockdown on OSCC progression. Overall, we demonstrated that OTUB1 might regulate OSCC progression by maintaining the stability of the RACK1 protein. These findings highlight the potential roles of the OTUB1/RACK1 axis as a potential therapeutic target in OSCC.

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation.

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

Loss of DJ-1 function contributes to Parkinson's disease pathogenesis in mice via RACK1-mediated PKC activation and MAO-B upregulation.

Parkinson's disease (PD) is a common neurodegenerative motor disorder characterized by a dramatic reduction in pars compacta of substantia nigra dopaminergic neurons and striatal dopamine (DA) levels. Mutations or deletions in the PARK7/DJ-1 gene are associated with an early-onset familial form of PD. DJ-1 protein prevents neurodegeneration via its regulation of oxidative stress and mitochondrial function as well as its roles in transcription and signal transduction. In this study, we investigated how loss of DJ-1 function affected DA degradation, ROS generation and mitochondrial dysfunction in neuronal cells. We showed that loss of DJ-1 significantly increased the expression of monoamine oxidase (MAO)-B but not MAO-A in both neuronal cells and primary astrocytes. In DJ-1-knockout (KO) mice, MAO-B protein levels in the substantia nigra (SN) and striatal regions were significantly increased. We demonstrated that the induction of MAO-B expression by DJ-1 deficiency depended on early growth response 1 (EGR1) in N2a cells. By coimmunoprecipitation omics analysis, we found that DJ-1 interacted with receptor of activated protein C kinase 1 (RACK1), a scaffolding protein, and thus inhibited the activity of the PKC/JNK/AP-1/EGR1 cascade. The PKC inhibitor sotrastaurin or the JNK inhibitor SP600125 completely inhibited DJ-1 deficiency-induced EGR1 and MAO-B expression in N2a cells. Moreover, the MAO-B inhibitor rasagiline inhibited mitochondrial ROS generation and rescued neuronal cell death caused by DJ-1 deficiency, especially in response to MPTP stimulation in vitro and in vivo. These results suggest that DJ-1 exerts neuroprotective effects by inhibiting the expression of MAO-B distributed at the mitochondrial outer membrane, which mediates DA degradation, ROS generation and mitochondrial dysfunction. This study reveals a mechanistic link between DJ-1 and MAO-B expression and contributes to understanding the crosslinks among pathogenic factors, mitochondrial dysfunction and oxidative stress in PD pathogenesis.

Scaffold protein RACK1A positively regulates leaf senescence by coordinating the EIN3-miR164-ORE1 transcriptional cascade in Arabidopsis.

Plants have adopted versatile scaffold proteins to facilitate the crosstalk between multiple signaling pathways. Leaf senescence is a well-programmed developmental stage that is coordinated by various external and internal signals. However, the functions of plant scaffold proteins in response to senescence signals are not well understood. Here, we report that the scaffold protein RACK1A (RECEPTOR FOR ACTIVATED C KINASE 1A) participates in leaf senescence mediated by ethylene signaling via the coordination of the EIN3-miR164-ORE1 transcriptional regulatory cascade. RACK1A is a novel positive regulator of ethylene-mediated leaf senescence. The rack1a mutant exhibits delayed leaf senescence, while transgenic lines overexpressing RACK1A display early leaf senescence. Moreover, RACK1A promotes EIN3 (ETHYLENE INSENSITIVE 3) protein accumulation, and directly interacts with EIN3 to enhance its DNA-binding activity. Together, they then associate with the miR164 promoter to inhibit its transcription, leading to the release of the inhibition on downstream ORE1 (ORESARA 1) transcription and the promotion of leaf senescence. This study reveals a mechanistic framework by which RACK1A promotes leaf senescence via the EIN3-miR164-ORE1 transcriptional cascade, and provides a paradigm for how scaffold proteins finely tune phytohormone signaling to control plant development.

CircVPRBP inhibits nodal metastasis of cervical cancer by impeding RACK1 O-GlcNAcylation and stability.

Lymph node (LN) metastasis is one of the most malignant clinical features in patients with cervical cancer (CCa). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with CCa. Circular RNAs (circRNA) play a critical role in the development of human cancers. However, the role and mechanism of circRNAs in lymph node metastasis remain largely unknown. Here, it is reported that loss expression of circRNA circVPRBP was closely associated with LN metastasis and poor survival of CCa patients. In vitro and in vivo assays showed that circVPRBP overexpression notably inhibited lymphangiogenesis and LN metastasis, whereas RfxCas13d mediated silencing of circVPRBP promoted lymphangiogenesis and the ability of the cervical cancer cells to metastasize to the LNs. Mechanistically, circVPRBP could bind to RACK1 and shield the S122 O-GlcNAcylation site to promote RACK1 degradation, resulting in inhibition of Galectin-1 mediated lymphangiogenesis and LN metastasis in CCa. Taken together, the results demonstrate that circVPRBP is a potential prognostic biomarker and a novel therapeutic target for LN metastasis in CCa patients.

Ribosomal RACK1 Regulates the Dendritic Arborization by Repressing FMRP Activity.

FMRP is an RNA-binding protein that represses the translation of specific mRNAs. In neurons, its depletion determines the exaggerated translation of mRNAs leading to dendritic and axonal aberrant development, two peculiar features of Fragile X syndrome patients. However, how FMRP binds to translational machinery to regulate the translation of its mRNA targets is not yet fully understood. Here, we show that FMRP localizes on translational machinery by interacting with the ribosomal binding protein, Receptor for Activated C Kinase 1 (RACK1). The binding of FMRP to RACK1 removes the translational repressive activity of FMRP and promotes the translation of PSD-95 mRNA, one specific target of FMRP. This binding also results in a reduction in the level of FMRP phosphorylation. We also find that the morphological abnormalities induced by Fmr1 siRNA in cortical neurons are rescued by the overexpression of a mutant form of RACK1 that cannot bind ribosomes. Thus, these results provide a new mechanism underlying FMRP activity that contributes to altered development in FXS. Moreover, these data confirm the role of ribosomal RACK1 as a ribosomal scaffold for RNA binding proteins.

Rack1 regulates cellular patterning and polarity in the mouse cochlea.

Rack1 features seven WD40 repeats that fold into a multifaceted scaffold used to build signaling complexes in a context-dependent manner. Previous in vitro studies have revealed associations between Rack1 and many other proteins. Rack 1 is required for establishing planar cell polarity (PCP) in zebrafish and Xenopus. However, any molecular role of Rack1 in protein complexes or polarity regulation remains unclear. Here, we show that Rack1 is an essential gene in mice. Conditional knockout of Rack1 shortened the cochlear duct and induced cellular patterning defects characteristic of defective convergent extension (this PCP process is mediated by cellular junctional remodeling in the developing cochlear epithelium). Also, cochlear hair cells were no longer uniformly oriented in Rack1 conditional knockout mutants. Rack1 was enriched in the cellular cortices of sensory hair cells. In Rack1-deficient cochleae, E-cadherin expression at the cellular boundaries was greatly reduced. Together, the findings reveal a molecular role of Rack1 in PCP signaling that likely involves modulation of E-cadherin levels at the adherens junctions of the plasma membrane.

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling.

Receptor for activated C kinase 1 (RACK1) is a small ribosomal subunit protein that is phosphorylated by vaccinia virus (VacV) to maximize translation of postreplicative (PR) mRNAs that harbor 5' polyA leaders. However, RACK1 is a multifunctional protein that both controls translation directly and acts as a scaffold for signaling to and from the ribosome. This includes stress signaling that is activated by ribosome-associated quality control (RQC) and ribotoxic stress response (RSR) pathways. As VacV infection activates RQC and stress signaling, whether RACK1 influences viral protein synthesis through its effects on translation, signaling, or both remains unclear. Examining the effects of genetic knockout of RACK1 on the phosphorylation of key mitogenic and stress-related kinases, we reveal that loss of RACK1 specifically blunts the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at late stages of infection. However, RACK1 was not required for JNK recruitment to ribosomes, and unlike RACK1 knockout, JNK inhibitors had no effect on viral protein synthesis. Moreover, reduced JNK activity during infection in RACK1 knockout cells contrasted with the absolute requirement for RACK1 in RSR-induced JNK phosphorylation. Comparing the effects of RACK1 knockout alongside inhibitors of late stage replication, our data suggest that JNK activation is only indirectly affected by the absence of RACK1 due to reduced viral protein accumulation. Cumulatively, our findings in the context of infection add further support for a model whereby RACK1 plays a specific and direct role in controlling translation of PR viral mRNAs that is independent of its role in ribosome-based stress signaling. IMPORTANCE Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal protein that regulates translation directly and mediates signaling to and from the ribosome. While recent work has shown that RACK1 is phosphorylated by vaccinia virus (VacV) to stimulate translation of postreplicative viral mRNAs, whether RACK1 also contributes to VacV replication through its roles in ribosome-based stress signaling remains unclear. Here, we characterize the role of RACK1 in infected cells. In doing so, we find that RACK1 is essential for stress signal activation by ribotoxic stress responses but not by VacV infection. Moreover, although the loss of RACK1 reduces the level of stress-associated JNK activation in infected cells, this is an indirect consequence of RACK1's specific requirement for the synthesis of postreplicative viral proteins, the accumulation of which determines the level of cellular stress. Our findings reveal both the specific role of RACK1 and the complex downstream effects of its control of viral protein synthesis in the context of infection.