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Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).

The ARF tumour suppressor stabilizes p53 by negatively regulating the E3 ubiquitin ligase MDM2 to promote cell cycle arrest and cell death. However, ARF is also able to arrest cell proliferation by inhibiting ribosome biogenesis. In greater part this is achieved by targeting the transcription termination factor I (TTF-I) for nucleolar export, leading to an inhibition of both ribosomal RNA synthesis and processing. We now show that in the absence of ARF, TTF-I is ubiquitinylated by MDM2. MDM2 interacts directly with TTF-I and regulates its cellular abundance by targeting it for degradation by the proteasome. Enhanced TTF-I levels inhibit ribosome biogenesis by suppressing ribosomal RNA synthesis and processing, strongly suggesting that exact TTF-I levels are critical for efficient ribosome biogenesis. We further show that concomitant with its ability to displace TTF-I from the nucleolus, ARF inhibits MDM2 ubiquitinylation of TTF-I by competitively binding to a site overlapping the MDM2 interaction site. Thus, both the sub-nuclear localization and the abundance of TTF-I are key regulators of ribosome biogenesis.

The p14/p19(ARF) (ARF) product of the CDKN2A gene displays tumor suppressor activity both in the presence and absence of p53/TP53. In p53-negative cells, ARF arrests cell proliferation, at least in part, by suppressing ribosomal RNA synthesis. We show that ARF does this by controlling the subnuclear localization of the RNA polymerase I transcription termination factor, TTF-I. TTF-I shuttles between nucleoplasm and nucleolus with the aid of the chaperone NPM/B23 and a nucleolar localization sequence within its N-terminal regulatory domain. ARF inhibits nucleolar import of TTF-I by binding to this nucleolar localization sequence, causing the accumulation of TTF-I in the nucleoplasm. Depletion of TTF-I recapitulates the effects of ARF on ribosomal RNA synthesis and is rescued by the introduction of a TTF-I transgene. Thus, our data delineate the pathway by which ARF regulates ribosomal RNA synthesis and provide a compelling explanation for the role of NPM., (Copyright 2010 Elsevier Inc. All rights reserved.)

The tumor suppressor p14/19ARF regulates ribosomal RNA (rRNA) synthesis by controlling the nucleolar localization of Transcription Termination Factor 1 (TTF1). However, the role played by TTF1 in regulating the rRNA genes and in potentially controlling growth has remained unclear. We now show that TTF1 expression regulates cell growth by determining the cellular complement of ribosomes. Unexpectedly, it achieves this by acting as a "roadblock" to synthesis of the noncoding LncRNA and pRNA that we show are generated from the "Spacer Promoter" duplications present upstream of the 47S pre‐rRNA promoter on the mouse and human ribosomal RNA genes. Unexpectedly, the endogenous generation of these noncoding RNAs does not induce CpG methylation or gene silencing. Rather, it acts in cis to suppress 47S preinitiation complex formation and hence de novo pre‐rRNA synthesis by a mechanism reminiscent of promoter interference or occlusion. Taken together, our data delineate a pathway from p19ARF to cell growth suppression via the regulation of ribosome biogenesis by noncoding RNAs and validate a key cellular growth law in mammalian cells. [ABSTRACT FROM AUTHOR]

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation. Author summary: The 200 Ribosomal RNA gene copies (the rDNA) account for 80% of total cellular RNA and determine the cell's ribosome complement and hence also its capacity to synthesize proteins. As a consequence, these genes directly control cell growth and their misfunction leads both to hypertrophic and developmental disorders. However, it is still unclear what determines the choice of which rDNA copies are activated and how this relates to the formation of the transcription preinitiation complex. Our study, based on the conditional deletion of TAF1B, a subunit of the TBP complex SL1, reveals how the DNA sequence non-specific HMGbox factor UBTF plays a sequence-specific role in SL1 recruitment and preinitiation complex formation. We further show how this role is degraded by a mutation that is the cause of the E210K recurrent pediatric neuroregression syndrome. The data suggest an induced-fit model of preinitiation complex formation in which UBTF creates a DNA folding that is specifically recognized by SL1. [ABSTRACT FROM AUTHOR]

Summary: Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to downregulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an explanation for the cytotoxic effects of loss of CpG methylation. [Copyright &y& Elsevier]