Instruct: Given a scientific claim, retrieve relevant passages that support or refute the claim.
Query: Pre-mRNAs associated with spliceosomal components are less stable than unassociated splicing substrates.
The development of in vitro assays to analyze pre-mRNA splicing resulted in the discovery of many fundamental features characterizing splicing signals and the machinery that completes this process. Because in vitro assays can be manipulated by various biochemical approaches, the versatility of investigating alternative pre-mRNA splicing in the test tube appears endless. Importantly, modifications in reaction conditions can lead to the accumulation, isolation, and characterization of reaction intermediates, a prerequisite for gaining mechanistic insights into how the spliceosome carries out intron removal, and how regulatory elements assist the general splicing machinery in defining splice sites and alternative exons. These considerable experimental advantages have made the in vitro splicing system a standard assay, even though this approach is independent from RNA transcription and other RNA processing events, and in some respects deviates from the natural process of mRNA biogenesis. Here, we describe the tools and techniques necessary to carry out in vitro splicing assays. Analyses of various experimental designs are presented to highlight the approaches taken to gain insights into the mechanisms by which splice site recognition and activation are communicated with the general splicing machinery. Methods to measure the kinetics of splicing, to observe the formation of the pre-spliceosomal complexes, and to manipulate and modify the in vitro system to resolve the regulatory influences in alternative splicing are presented.
Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.
Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.
3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3'-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II.
Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.
Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs (miRNAs) from pre-miRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage and an N-terminal helicase motif, whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.
The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. Here, we show, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. We therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference.
Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.
In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.
SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes.
RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression.
Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process, and factors involved remain largely unknown. Here, we have identified the DEAD-box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability or 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis, and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that takes place mainly near the mitochondrial nucleoids, in the compartment defined by the RNA granules.
Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.
Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures "mitochondrial RNA granules. "
Many cellular proteins perform their roles within macromolecular assemblies. Hence, an understanding of how these multiprotein complexes form is a fundamental question in cell biology. We developed a translation-controlled pulse-chase system that allows time-resolved isolation of newly forming multiprotein complexes in chemical quantities suitable for biochemical and cell biological analysis. The "pulse" is triggered by an unnatural amino acid, which induces immediate translation of an amber stop codon repressed mRNA encoding the protein of interest with a built-in tag for detection and purification. The "chase" is elicited by stopping translation of this bait via a riboswitch in the respective mRNA. Over the course of validating our method, we discovered a distinct time-resolved assembly step during NPC biogenesis and could directly monitor the spatiotemporal maturation of preribosomes via immunofluorescence detection and purification of a pulse-labeled ribosomal protein. Thus, we provide an innovative strategy to study dynamic protein assembly within cellular networks.
Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.
Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.
HEN1-mediated 2'-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3' terminal uridylation [1-8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3' end of RNA, which is completely abolished by 2'-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3'-to-5' exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals.
Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.
The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.
Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.
RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.
The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.