Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae.

Ribosome biogenesis is a multi-step process that couples cell growth with cell proliferation. Although several large-scale analysis of pre-ribosomal particles have identified numerous trans-acting factors involved in this process, many proteins involved in pre-rRNA processing and ribosomal subunit maturation have yet to be identified. Las1 was originally identified in Saccharomyces cerevisiae as a protein involved in cell morphogenesis. We previously demonstrated that the human homolog, Las1L, is required for efficient ITS2 rRNA processing and synthesis of the 60S ribosomal subunit. Here, we report that the functions of Las1 in ribosome biogenesis are also conserved in S. cerevisiae. Depletion of Las1 led to the accumulation of both the 27S and 7S rRNA intermediates and impaired the synthesis of the 60S subunit. We show that Las1 co-precipitates mainly with the 27S rRNA and associates with an Nsa1 and Rix1-containing pre-60S particle. We further identify Grc3 as a major Las1-interacting protein. We demonstrate that the kinase activity of Grc3 is required for efficient pre-rRNA processing and that depletion of Grc3 leads to rRNA processing defects similar to the ones observed in Las1-depleted cells. We propose that Las1 and Grc3 function together in a conserved mechanism to modulate rRNA processing and eukaryotic ribosome biogenesis.

The impact of ribosome biogenesis in cancer: from proliferation to metastasis.

The dysregulation of ribosome biogenesis is a hallmark of cancer, facilitating the adaptation to altered translational demands essential for various aspects of tumor progression. This review explores the intricate interplay between ribosome biogenesis and cancer development, highlighting dynamic regulation orchestrated by key oncogenic signaling pathways. Recent studies reveal the multifaceted roles of ribosomes, extending beyond protein factories to include regulatory functions in mRNA translation. Dysregulated ribosome biogenesis not only hampers precise control of global protein production and proliferation but also influences processes such as the maintenance of stem cell-like properties and epithelial-mesenchymal transition, contributing to cancer progression. Interference with ribosome biogenesis, notably through RNA Pol I inhibition, elicits a stress response marked by nucleolar integrity loss, and subsequent G1-cell cycle arrest or cell death. These findings suggest that cancer cells may rely on heightened RNA Pol I transcription, rendering ribosomal RNA synthesis a potential therapeutic vulnerability. The review further explores targeting ribosome biogenesis vulnerabilities as a promising strategy to disrupt global ribosome production, presenting therapeutic opportunities for cancer treatment.

p53 induces a survival transcriptional response after nucleolar stress.

Accumulating evidence indicates that increased ribosome biogenesis is a hallmark of cancer. It is well established that inhibition of any steps of ribosome biogenesis induces nucleolar stress characterized by p53 activation and subsequent cell cycle arrest and/or cell death. However, cells derived from solid tumors have demonstrated different degrees of sensitivity to ribosome biogenesis inhibition, where cytostatic effects rather than apoptosis are observed. The reason for this is not clear, and the p53-specific transcriptional program induced after nucleolar stress has not been previously investigated. Here we demonstrate that blocking rRNA synthesis by depletion of essential rRNA processing factors such as LAS1L, PELP1, and NOP2 or by inhibition of RNA Pol I with the specific small molecule inhibitor CX-5461, mainly induce cell cycle arrest accompanied by autophagy in solid tumor-derived cell lines. Using gene expression analysis, we find that p53 orchestrates a transcriptional program involved in promoting metabolic remodeling and autophagy to help cells survive under nucleolar stress. Importantly, our study demonstrates that blocking autophagy significantly sensitizes cancer cells to RNA Pol I inhibition by CX-5461, suggesting that interfering with autophagy should be considered a strategy to heighten the responsiveness of ribosome biogenesis-targeted therapies in p53-positive tumors.

p53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression of MCOLN1.

Inhibition of TRPML1, which is encoded by MCOLN1, is known to deter cell proliferation in various malignancies. Here, we report that the tumor suppressor, p53, represses MCOLN1 in the urothelium such that either the constitutive loss or ectopic knockdown of TP53-in both healthy and bladder cancer cells-increased MCOLN1 expression. Conversely, nutlin-mediated activation of p53 led to the repression of MCOLN1. Elevated MCOLN1 expression in p53-deficient cancer cells, though not sufficient for bolstering proliferation, augmented the effects of oncogenic HRAS on proliferation, cytokine production, and invasion. Our data suggest that owing to derepression of MCOLN1, urothelial cells lacking p53 are poised for tumorigenesis driven by oncogenic HRAS. Given our prior findings that HRAS mutations predict addiction to TRPML1, this study points to the utility of TRPML1 inhibitors for mitigating the growth of a subset of urothelial tumors that lack p53.

Relocalized p27Kip1 tumor suppressor functions as a cytoplasmic metastatic oncogene in melanoma.

The p27 tumor suppressor negatively regulates G1 cell cycle progression. However, human malignancies rarely select for deletion/inactivation of p27, a hallmark of tumor suppressor genes. Instead, p27 is degraded or relocalized to the cytoplasm in aggressive malignancies, supporting the notion that p27 sequestration from its nuclear cyclin:cyclin-dependent kinase (cdk) targets is critical. However, emerging cell biology data suggest a novel cdk-independent cytoplasmic function of p27 in cell migration. Here, we find cytoplasmic p27 in 70% of invasive and metastatic melanomas. In contrast, no cytoplasmic p27 was detected in noninvasive, basement membrane-confined melanoma in situ, suggesting a late oncogenic role for cytoplasmic p27 in metastasis. Targeted cytoplasmic expression of wild-type or non-cdk-binding p27 at subphysiologic levels induced melanoma motility and resulted in numerous metastases to lymph node, lung, and peritoneum. These observations point to a prominent role of cytoplasmic p27 in metastatic disease that is independent of cyclin:cdk regulation or mere nuclear loss.

Thiol stress-dependent aggregation of the glycolytic enzyme triose phosphate isomerase in yeast and human cells.

The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants, and xenobiotics. Cysteine-containing proteins are especially at risk, as the thiol side chain is subject to oxidation, adduction, and chelation by thiol-reactive compounds. The thiol-chelating heavy metal cadmium is a highly toxic environmental pollutant demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of cadmium toxicity responsible for these outcomes are largely unknown. Using fluorescent protein fusion to cytosolic proteins with known redox-active cysteines, we identified the yeast glycolytic enzyme triose phosphate isomerase as being aggregation-prone in response to cadmium and to glucose depletion in chronologically aging cultures. Cadmium-induced aggregation was limited to newly synthesized Tpi1 that was recruited to foci containing the disaggregase Hsp104 and the peroxiredoxin chaperone Tsa1. Misfolding of nascent Tpi1 in response to both cadmium and glucose-depletion stress required both cysteines, implying that thiol status in this protein directly influences folding. We also demonstrate that cadmium proteotoxicity is conserved between yeast and human cells, as HEK293 and HCT116 cell lines exhibit recruitment of the protein chaperone Hsp70 to visible foci. Moreover, human TPI, mutations in which cause a glycolytic deficiency syndrome, also forms aggregates in response to cadmium treatment, suggesting that this conserved enzyme is folding-labile and may be a useful endogenous model for investigating thiol-specific proteotoxicity.

Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides.

Protein transduction domains (PTDs), such as the TAT PTD, have been shown to deliver a wide variety of cargo in cell culture and to treat preclinical models of cancer and cerebral ischemia. The TAT PTD enters cells by a lipid raft-dependent macropinocytosis mechanism that all cells perform. Consequently, PTDs resemble small-molecule therapeutics in their lack of pharmacologic tissue specificity in vivo. However, several human malignancies overexpress specific receptors, including HER2 in breast cancer, GnRH in ovarian carcinomas, and CXC chemokine receptor 4 (CXCR4) in multiple malignancies. To target tumor cells that overexpress the CXCR4 receptor, we linked the CXCR4 DV3 ligand to two transducible anticancer peptides: a p53-activating peptide (DV3-TATp53C') and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL). Treatment of tumor cells expressing the CXCR4 receptor with either the DV3-TATp53C' or DV3-TAT-RxL targeted peptides resulted in an enhancement of tumor cell killing compared with treatment with nontargeted parental peptides. In contrast, there was no difference between DV3 targeted peptide and nontargeted, parental peptide treatment of non-CXCR4-expressing tumor cells. These observations show that a multidomain approach can be used to further refine and enhance the tumor selectivity of biologically active, transducible macromolecules for treating cancer.

p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress.

The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks.

Protein transduction technology offers novel therapeutic approach for brain ischemia.

Transient or permanent reduction in cerebral blood flow following ischemia can lead to severe and irreversible tissue damage to the brain. Emerging biochemical evidence suggests a role for apoptosis in neuronal death following cerebral ischemia. Despite the abundance of studies on the subject, therapeutic interventions for ischemia-related cell injury have so far proved disappointing in clinical trials. Recently, four new, exciting studies reported the use of protein transduction technology to deliver anti-apoptotic molecules to protect neuronal cells following ischemic stroke in vivo. These studies offer new avenues for the treatment and prevention of cell death following brain injuries.

LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis.

The coordination of RNA polymerase I transcription with pre-rRNA processing, preribosomal particle assembly, and nuclear export is a finely tuned process requiring the concerted actions of a number of accessory factors. However, the exact functions of some of these proteins and how they assemble in subcomplexes remain poorly defined. LAS1L was first described as a nucleolar protein required for maturation of the 60S preribosomal subunit. In this paper, we demonstrate that LAS1L interacts with PELP1, TEX10, and WDR18, the mammalian homologues of the budding yeast Rix1 complex, along with NOL9 and SENP3, to form a novel nucleolar complex that cofractionates with the 60S preribosomal subunit. Depletion of LAS1L-associated proteins results in a p53-dependent G1 arrest and leads to defects in processing of the pre-rRNA internal transcribed spacer 2 region. We further show that the nucleolar localization of this complex requires active RNA polymerase I transcription and the small ubiquitin-like modifier-specific protease SENP3. Taken together, our data identify a novel mammalian complex required for 60S ribosomal subunit synthesis, providing further insight into the intricate, yet poorly described, process of ribosome biogenesis in higher eukaryotes.

Las1L is a nucleolar protein required for cell proliferation and ribosome biogenesis.

Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.

Gris1, a new common integration site in Graffi murine leukemia virus-induced leukemias: overexpression of a truncated cyclin D2 due to alternative splicing.

The Graffi murine leukemia virus is a nondefective ecotropic retrovirus that was originally reported to induce myeloid leukemia in some strains of mice (A. Graffi, Ann. N.Y. Acad. Sci. 68:540-558, 1957). Using provirus-flanking sequences as DNA probes, we identified a new common retroviral integration site called Gris1 (for Graffi integration site 1). Viral integrations in Gris1 were detected in 13% of the tumors analyzed. The Gris1 locus was mapped to the distal region of mouse chromosome 6, 85 kb upstream of the cyclin D2 gene. Such viral integration in Gris1 causes overexpression of the normal 6.5-kb major transcript of cyclin D2 but also induces the expression of a new, alternatively spliced 1.1-kb transcript from the cyclin D2 gene that encodes a truncated cyclin D2 of 17 kDa. The expression of this 1.1-kb transcript is specific to tumors in which Gris1 is rearranged but is also detected at low levels in normal tissue.

Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-dependent kinases 4/6 activity in melanocyte survival.

Deregulation of the p16INK4a-cyclin D:cyclin-dependent kinases (cdk) 4/6-retinoblastoma (pRB) pathway is a common paradigm in the oncogenic transformation of human cells and suggests that this pathway functions linearly in malignant transformation. However, it is not understood why p16INK4a and cyclin D:cdk4/6 mutations are disproportionately more common than the rare genetic event of RB inactivation in human malignancies such as melanoma. To better understand how these complexes contribute to altered tissue homeostasis, we blocked cdk4/6 activation and acutely inactivated Rb by conditional mutagenesis during mouse hair follicle cycling. Inhibition of cdk4/6 in the skin by subcutaneous administration of a membrane-transducible TAT-p16INK4a protein completely blocked hair follicle growth and differentiation. In contrast, acute disruption of Rb in the skin of homozygous RbLoxP/LoxP mice via subcutaneous administration of TAT-Cre recombinase failed to affect hair growth. However, loss of Rb resulted in severe depigmentation of hair follicles. Further analysis of follicular melanocytes in vivo and in primary cell culture demonstrated that pRB plays a cell-autonomous role in melanocyte survival. Moreover, functional inactivation of all three Rb family members (Rb, p107, and p130) in primary melanocytes by treatment with a transducible TAT-E1A protein did not rescue the apoptotic phenotype. These findings suggest that deregulated cyclin D:cdk4/6 complexes and pRB perform nonoverlapping functions in vivo and provide a cellular mechanism that accounts for the low incidence of RB inactivation in cancers such as melanoma.