A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.

Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.

U2AF2-SNORA68 promotes triple-negative breast cancer stemness through the translocation of RPL23 from nucleoplasm to nucleolus and c-Myc expression.

BACKGROUND: Small nucleolar RNAs (snoRNAs) play key roles in ribosome biosynthesis. However, the mechanism by which snoRNAs regulate cancer stemness remains to be fully elucidated. METHODS: SNORA68 expression was evaluated in breast cancer tissues by in situ hybridization and qRT‒PCR. Proliferation, migration, apoptosis and stemness analyses were used to determine the role of SNORA68 in carcinogenesis and stemness maintenance. Mechanistically, RNA pull-down, RNA immunoprecipitation (RIP), cell fractionation and coimmunoprecipitation assays were conducted. RESULTS: SNORA68 exhibited high expression in triple-negative breast cancer (TNBC) and was significantly correlated with tumor size (P = 0.048), ki-67 level (P = 0.037), and TNM stage (P = 0.015). The plasma SNORA68 concentration was significantly lower in patients who achieved clinical benefit. The SNORA68-high patients had significantly shorter disease-free survival (DFS) (P = 0.036). Functionally, SNORA68 was found to promote the cell stemness and carcinogenesis of TNBC in vitro and in vivo. Furthermore, elevated SNORA68 expression led to increased nucleolar RPL23 expression and retained RPL23 in the nucleolus by binding U2AF2. RPL23 in the nucleolus subsequently upregulated c-Myc expression. This pathway was validated using a xenograft model. CONCLUSION: U2AF2-SNORA68 promotes TNBC stemness by retaining RPL23 in the nucleolus and increasing c-Myc expression, which provides new insight into the regulatory mechanism of stemness.

Biological role and diagnostic utility of ribosomal protein L23a pseudogene 53 in cutaneous melanoma.

Background: Skin melanoma is one of the deadliest types of skin cancer and develops from melanocytes. The genetic aberrations in protein-coding genes are well characterized, but little is known about changes in non-coding RNAs (ncRNAs) such as pseudogenes. Ribosomal protein pseudogenes (RPPs) have been described as the largest group of pseudogenes which are dispersed in the human genome. Materials and methids: We looked deeply at the role of one of them, ribosomal protein L23a pseudogene 53 (RPL23AP53), and its potential diagnostic use. The expression level of RPL23AP53 was profiled in melanoma cell lines using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and analyzed based on the Cancer Genome Atlas (TCGA) data depending on BRAF status and clinicopathological parameters. Cellular phenotype, which was associated with RPL23AP53 levels, was described based on the REACTOME pathway browser, Gene Set Enrichment Analysis (GSEA) analysis as well as Immune and ESTIMATE Scores. Results: We indicted in vitro changes in RPL23AP53 level depending on a cell line, and based on in silico analysis of TCGA samples demonstrated significant differences in RPL23AP53 expression between primary and metastatic melanoma, as well as correlation between RPL23AP53 and overall survival. No differences depending on BRAF status were observed. RPL23AP53 is associated with several signaling pathways and cellular processes. Conclusions: This study showed that patients with higher expression of RPL23AP53 displayed changed infiltration of lymphocytes, macrophages, and neutrophils compared to groups with lower expression of RPL23AP53. RPL23AP53 pseudogene is differently expressed in melanoma compared with normal tissue and its expression is associated with cellular proliferation. Thus, it may be considered as an indicator of patients' survival and a marker for the immune profile assessment.

Circ_RPL23A acts as a miR-1233 sponge to suppress the progression of clear cell renal cell carcinoma by promoting ACAT2.

Clear cell renal cell carcinoma (ccRCC) is a prevalent urological carcinoma with high metastatic risk. Circular RNAs (circRNAs) have been identified as effective diagnostic and therapeutic biomarkers for ccRCC. This research aims to disclose the effect and regulatory mechanism of circRNA ribosomal protein L23a (circ_RPL23A) in ccRCC. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to examine circ_RPL23A, microRNA-1233 (miR-1233) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). Cell cycle progression, apoptosis, cell viability, invasion and migration, which were respectively conducted by using flow cytometry, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), transwell assays. The levels of ACAT2 protein and cell cycle proteins, proliferation-associated protein, and epithelial-mesenchymal transition (EMT) associated proteins were measured by western blot. Target relationship was analyzed via dual-luciferase reporter assay and RNA pull down assay. The animal model was used to study how circ_RPL23A affects in vivo. Circ_RPL23A was lower expressed in ccRCC tissues and cells. The elevated circ_RPL23A suppressed cell cycle progression, proliferation, migration and invasion but promoted apoptosis in ccRCC cells. MiR-1233 was a target of circ_RPL23A and direct targeted to ACAT2. Besides, circ_RPL23A exerted its anti-tumor effect by sponging miR-1233, and then relieved the inhibition effect of miR-1233 on ACAT2. Overexpression of circ_RPL23A also curbed ccRCC tumor growth in vivo. Circ_RPL23A inhibited ccRCC progression by upregulating ACAT2 expression by competitively binding miR-1233, which might provide an in-depth cognition for ccRCC pathogenesis and circ_RPL23A might be a promising biomarker in ccRCC diagnosis and treatment.

GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system.

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.

Arabidopsis paralogous genes RPL23aA and RPL23aB encode functionally equivalent proteins.

BACKGROUND: In plants, each ribosomal protein (RP) is encoded by a small gene family but it is largely unknown whether the family members are functionally diversified. There are two RPL23a paralogous genes (RPL23aA and RPL23aB) encoding cytoplasmic ribosomal proteins in Arabidopsis thaliana. Knock-down of RPL23aA using RNAi impeded growth and led to morphological abnormalities, whereas knock-out of RPL23aB had no observable phenotype, thus these two RPL23a paralogous proteins have been used as examples of ribosomal protein paralogues with functional divergence in many published papers. RESULTS: In this study, we characterized T-DNA insertion mutants of RPL23aA and RPL23aB. A rare non-allelic non-complementation phenomenon was found in the F1 progeny of the rpl23aa X rpl23ab cross, which revealed a dosage effect of these two genes. Both RPL23aA and RPL23aB were found to be expressed almost in all examined tissues as revealed by GUS reporter analysis. Expression of RPL23aB driven by the RPL23aA promoter can rescue the phenotype of rpl23aa, indicating these two proteins are actually equivalent in function. Interestingly, based on the publicly available RNA-seq data, we found that these two RPL23a paralogues were expressed in a concerted manner and the expression level of RPL23aA was much higher than that of RPL23aB at different developmental stages and in different tissues. CONCLUSIONS: Our findings suggest that the two RPL23a paralogous proteins are functionally equivalent but the two genes are not. RPL23aA plays a predominant role due to its higher expression levels. RPL23aB plays a lesser role due to its lower expression. The presence of paralogous genes for the RPL23a protein in plants might be necessary to maintain its adequate dosage.

Loss of FBXO7 results in a Parkinson's-like dopaminergic degeneration via an RPL23-MDM2-TP53 pathway.

The field of Parkinson's disease research has been impeded by the absence of animal models that clearly phenocopy the features of this neurodegenerative condition. Mutations in FBXO7/PARK15 are associated with both sporadic Parkinson's disease and a severe form of autosomal recessive early-onset Parkinsonism. Here we report that conditional deletion of Fbxo7 in the midbrain dopamine neurons results in an early reduction in striatal dopamine levels, together with a slow, progressive loss of midbrain dopamine neurons and onset of locomotor defects. Unexpectedly, a later compensatory response led to a near-full restoration of dopaminergic fibre innervation in the striatum, but nigral cell loss was irreversible. Mechanistically, there was increased expression in the dopamine neurons of FBXO7-interacting protein, RPL23, which is a sensor of ribosomal stress that inhibits MDM2, the negative regulator of p53. A corresponding activated p53 transcriptional signature biased towards pro-apoptotic genes was also observed. These data suggest that the neuroprotective role of FBXO7 involves its suppression of the RPL23-MDM2-p53 axis that promotes cell death in dopaminergic midbrain neurons. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Corrigendum: Ribosomal protein L23 drives the metastasis of hepatocellular carcinoma via upregulating MMP9.

[This corrects the article DOI: 10.3389/fonc.2021.779748.].

cDNA cloning, overexpression, purification and pharmacologic evaluation for anticancer activity of ribosomal protein L23A gene (RPL23A) from the Giant Panda.

RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A) gene of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF) of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 µg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids orientation for the research and development of cancer protein drugs as well as possible anti-cancer mechanisms. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL23A responsible for its anticancer activity.

Targeting RPL23 restores chemosensitivity of cisplatin-resistant ovarian carcinoma by inhibiting EMT.

Chemoresistance is the inevitable outcome of chemotherapy for epithelial ovarian carcinoma (EOC), and its mechanism is still not fully understood. This study explored the role of ribosomal protein L23 (RPL23) in cisplatin resistance of EOC. WGCNA based on TCGA and GEO was used to screen and analyze target genes related to EOC chemotherapy sensitivity. Clinical samples of cisplatin resistance were collected to detect the expression of target genes. Cisplatin resistance was induced in EOC cell lines A2780 and SKOV3. The cell abilities of invasion, migration and adhesion were observed. Western blotting was used to detect protein expressions. Bioinformatics analysis showed that RPL23 may be related to EOC chemotherapy sensitivity, and was highly expressed in clinical samples and cell lines of cisplatin-resistant. After A2780 and SKOV3 were resistant to cisplatin, the inhibitory abilities of therapeutic dose of cisplatin on their invasion, migration and adhesion were significantly attenuated, and N-cadherin and vimentin were significantly up-regulated while E-cadherin was significantly down-regulated. However, above phenomena were significantly reversed after RPL23 knockdown. Taken together, the overexpressed RPL23 may lead to platinum resistance by inducing epithelial-mesenchymal transition (EMT) in EOC. Targeting knockdown RPL23 would restore the sensitivity of EOC cells to cisplatin by inhibiting EMT, suggesting that RPL23 is a potential therapeutic target for EOC after platinum resistance.

Novel insights into the mechanisms by which lncRNA HOTAIR regulates migration and invasion in HeLa cells.

HOTAIR, as one of the few well-studied oncogenic lncRNAs, is involved in human tumorigenesis and is dys-regulated in most human cancers. The transcription co-activator factor YAP1 is broadly expressed in many tissues, and promotes cancer metastasis and progression. However, the precise biological roles of HOTAIR and YAP1 in cancer cells remain unclear. In this study, we showed that HOTAIR regulates H3K27 histone modification in the promoter of miR-200a to mediate miR-200a expression by recruiting EZH2. YAP1, as a potential target gene of miR-200a, aggravated the effects of miR-200a on the migration and invasion of HeLa cells. YAP1 activated the transcription of RPL23, which is a novel downstream transcriptional-regulator of YAP1. Agreement with this, the expression of YAP1 and RPL23 was dramatically decreased after injecting HeLa cells transfected with siHOTAIR in a xenograft mouse model. Accordingly, we propose a novel model of the molecular mechanism by which HOTAIR promotes the migration and invasion of cancer cells involving the miR-200a-3p/YAP1/RPL23 axis.

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes.

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of 'common cause' attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

Ribosomal Protein L23 Drives the Metastasis of Hepatocellular Carcinoma via Upregulating MMP9.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. Tumor metastasis is one of the major causes of high mortality of HCC. Identifying underlying key factors contributing to invasion and metastasis is critical to understand the molecular mechanisms of HCC metastasis. Here, we identified RNA binding protein L23 (RPL23) as a tumor metastasis driver in HCC. RPL23 was significantly upregulated in HCC tissues compared to adjacent normal tissues, and closely related to poor clinical outcomes in HCC patients. RPL23 depletion inhibited HCC cell proliferation, migration and invasion, and distant metastasis. Mechanistically, RPL23 directly associated with 3'UTR of MMP9, therefore positively regulated MMP9 expression. In conclusion, we identified that RPL23 might play an important role in HCC metastasis in an MMP9-dependent manner and be a potential therapeutic target for HCC tumorigenesis and metastasis.

Expression Profiling of Ribosomal Protein Gene Family in Dehydration Stress Responses and Characterization of Transgenic Rice Plants Overexpressing RPL23A for Water-Use Efficiency and Tolerance to Drought and Salt Stresses.

Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL) subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE) with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a). In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP) gene family including both small (RPS) and large (RPL) subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24, and 31 and RPS4, 10 and 18a. To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ13C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt stresses. Taken together, our findings provide a secure basis for the RPL gene family expression as a potential resource for exploring abiotic stress tolerant properties in rice.

The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14.

BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPß, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPß. Depletion of BCCIPß reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPß leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPß functions as nuclear chaperone for RPL23.

Cloning and characterization of a ribosomal protein L23a gene from Small Tail Han sheep by screening of a cDNA expression library.

As an indispensable component of the eukaryotic ribosome, ribosomal protein L23a plays an important role in protein synthesis, folding and sorting. In this study, the cDNA fragment of ribosomal protein L23a with 471 bp in size was screened from the Small Tail Han sheep ear marginal tissue cDNA expression library, it has 157 amino acids and a molecular weight of 17.69 kDa. The nucleotide sequence of L23a shares a high homology with those of human, mouse, cattle and pig of 91.51%, 88.32%, 96.18% and 93.84%, respectively. L23a is highly basic, containing a combined 45 Arg, Lys, and His residues and only 14 Asp and Glu residues. The expression pattern and intra-cellular distribution of recombinant L23a proteins in Ujumqin sheep fibroblast cells were analyzed after transfected with the plasmid pEGFP-N3-RPL23A, there were green fluorescence signals both in the cytoplasm and nucleolus of transfected cells after 24 h, the number of positive cells was increased with time, and they reached the peak level after 48 h of transfection. The transfection efficiency was 22.8%. Expression patterns of recombinant L23a gene in Escherichia coli were different with induction temperature, inductor concentration and induction time, when the IPTG concentration was 0.1 mmol/L and induction temperature was 37°, L23a protein expression was increased with induction time.

Construction and screening of RPL23-siRNA interference fragments / 实用肿瘤学杂志

Ob jective To construct and screen out the RPL 23-siRNA interference fragments ,providing the basis for the following experiments about the correlation with RPL 23 and gastric cancer .Methods The RPL23-siRNA,synthesized chemically through lipofection ,were selected from three target sequences by RNA in-terference and detected by real -time PCR and Western blot .Results Compared with normal cell group and RPL23 control group ,the mRNA and protein expression of RPL 23 in the other 3 interference groups were signifi-cantly decreased(P<0.01).Multiple comparisons showed that the interference efficiency of RPL 23 -siRNA1 group was significantly higher than that of RPL 23-siRNA2 group and RPL23-siRNA3 group(P<0.01).Con-clusion The RPL23-siRNA interference fragment can be successfully constructed and screened out ,which pro-vides the basis for the following experiments .