Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2'-O-methylation.

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.

Three-Dimensional CeO2 Woodpile Nanostructures To Enhance Performance of Enzymatic Glucose Biosensors.

Fabrication of detection elements with ultrahigh surface area is essential for improving the sensitivity of analyte detection. Here, we report a direct patterning technique to fabricate three-dimensional CeO2 nanoelectrode arrays for biosensor application over relatively large areas. The fabrication approach, which employs nanoimprint lithography and a CeO2 nanoparticle-based ink, enables the direct, high-throughput patterning of nanostructures and is scalable, integrable, and of low cost. With the convenience of sequential imprinting, multilayered woodpile nanostructures with prescribed numbers of layers were achieved in a "stacked-up" architecture and were successfully fabricated over large areas. To demonstrate application as a biosensor, an enzymatic glucose sensor was developed. The sensitivity of glucose sensors can be enhanced simply by increasing the number of layers, which multiplies surface area while maintaining a constant footprint. The four-layer woodpile nanostructure of CeO2 glucose sensor exhibited enhanced sensitivity (42.8 µA mM-1 cm-2) and good selectivity. This direct imprinting strategy for three-dimensional sensing architectures is potentially extendable to other electroactive materials and other sensing applications.

The ribosome biogenesis factor yUtp23/hUTP23 coordinates key interactions in the yeast and human pre-40S particle and hUTP23 contains an essential PIN domain.

Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA-protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3΄ half of the snR30 snoRNA. Protein-protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation.

The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans.

During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.

The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21.

Translation fidelity and efficiency require multiple ribosomal (r)RNA modifications that are mostly mediated by small nucleolar (sno)RNPs during ribosome production. Overlapping basepairing of snoRNAs with pre-rRNAs often necessitates sequential and efficient association and dissociation of the snoRNPs, however, how such hierarchy is established has remained unknown so far. Here, we identify several late-acting snoRNAs that bind pre-40S particles in human cells and show that their association and function in pre-40S complexes is regulated by the RNA helicase DDX21. We map DDX21 crosslinking sites on pre-rRNAs and show their overlap with the basepairing sites of the affected snoRNAs. While DDX21 activity is required for recruitment of the late-acting snoRNAs SNORD56 and SNORD68, earlier snoRNAs are not affected by DDX21 depletion. Together, these observations provide an understanding of the timing and ordered hierarchy of snoRNP action in pre-40S maturation and reveal a novel mode of regulation of snoRNP function by an RNA helicase in human cells.

Integrity of SRP RNA is ensured by La and the nuclear RNA quality control machinery.

The RNA component of signal recognition particle (SRP) is transcribed by RNA polymerase III, and most steps in SRP biogenesis occur in the nucleolus. Here, we examine processing and quality control of the yeast SRP RNA (scR1). In common with other pol III transcripts, scR1 terminates in a U-tract, and mature scR1 retains a U4-5 sequence at its 3' end. In cells lacking the exonuclease Rex1, scR1 terminates in a longer U5-6 tail that presumably represents the primary transcript. The 3' U-tract of scR1 is protected from aberrant processing by the La homologue, Lhp1 and overexpressed Lhp1 apparently competes with both the RNA surveillance system and SRP assembly factors. Unexpectedly, the TRAMP and exosome nuclear RNA surveillance complexes are also implicated in protecting the 3' end of scR1, which accumulates in the nucleolus of cells lacking the activities of these complexes. Misassembled scR1 has a primary degradation pathway in which Rrp6 acts early, followed by TRAMP-stimulated exonuclease degradation by the exosome. We conclude that the RNA surveillance machinery has key roles in both SRP biogenesis and quality control of the RNA, potentially facilitating the decision between these alternative fates.

The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA.

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5' external transcribed spacer (5'ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5'ETS, at site A', upstream of A0, has also been reported. Here, we have investigated how A' processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A' cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A' cleavage in humans. A' cleavage is largely bypassed when XRN2 is depleted, and we also discover that A' cleavage is not always the initial processing event in all cell types. Together, our data suggest that A' cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells.

The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress.

Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production.

Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing.

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3' to 5' exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference-mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits.

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.

Comparison of the yeast and human nuclear exosome complexes.

Most RNAs in eukaryotic cells are produced as precursors that undergo processing at the 3' and/or 5' end to generate the mature transcript. In addition, many transcripts are degraded not only as part of normal recycling, but also when recognized as aberrant by the RNA surveillance machinery. The exosome, a conserved multiprotein complex containing two nucleases, is involved in both the 3' processing and the turnover of many RNAs in the cell. A series of factors, including the TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex, Mpp6 and Rrp47, help to define the targets to be processed and/or degraded and assist in exosome function. The majority of the data on the exosome and RNA maturation/decay have been derived from work performed in the yeast Saccharomyces cerevisiae. In the present paper, we provide an overview of the exosome and its role in RNA processing/degradation and discuss important new insights into exosome composition and function in human cells.

The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA.

Box C/D and H/ACA RNPs are essential ribonucleoprotein particles that are found throughout both eukaryotes [small nucleolar RNPs (snoRNPs)] and archaea [snoRNP-like complexes (sRNPs)]. These complexes catalyze the site-specific pseudouridylation and most of the methylation of ribosomal RNA (rRNA). The numerous modifications, which are clustered in functionally important regions of the rRNA, are important for rRNA folding and ribosome function. The RNA component of the complexes [small nucleolar RNA (snoRNA) or small RNA (sRNA)] functions in substrate binding by base pairing with the target site and as a scaffold coordinating the organization of the complex. In eukaryotes, a subset of snoRNPs do not catalyze modification but, through base pairing to the rRNA or flanking precursor sequences, direct pre-rRNA folding and are essential for rRNA processing. In the last few years there have been significant advances in our understanding of the structure of archaeal sRNPs. High resolution structures of the archaeal C/D and H/ACA sRNPs have not only provided a detailed understanding of the molecular architecture of these complexes but also produced key insights into substrate binding and product release. In both cases, this is mediated by significant movement in the complexes. Advances have also been made in our knowledge of snoRNP recruitment and release from pre-ribosome complexes in eukaryotes. New snoRNA-rRNA interactions have been documented, and the roles of RNA helicases in releasing snoRNP complexes from the rRNA have been described.

The spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of the eukaryotic box C/D snoRNP nucleotide modification complex.

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.

A novel Nop5-sRNA interaction that is required for efficient archaeal box C/D sRNP formation.

Archaeal and eukaryotic box C/D RNPs catalyze the 2'-O-methylation of ribosomal RNA, a modification that is essential for the correct folding and function of the ribosome. Each archaeal RNP contains three core proteins--L7Ae, Nop5, and fibrillarin (methyltransferase)--and a box C/D sRNA. Base-pairing between the sRNA guide region and the rRNA directs target site selection with the C/D and related C'/D' motifs functioning as protein binding sites. Recent structural analysis of in vitro assembled archaeal complexes has produced two divergent models of box C/D sRNP structure. In one model, the complex is proposed to be monomeric, while the other suggests a dimeric sRNP. The position of the RNA in the RNP is significantly different in each model. We have used UV-cross-linking to characterize protein-RNA contacts in the in vitro assembled Pyrococcus furiosus box C/D sRNP. The P. furiosus sRNP components assemble into complexes that are the expected size of di-sRNPs. Analysis of UV-induced protein-RNA cross-links revealed a novel interaction between the ALFR motif, in the Nop domain of Nop5, and the guide/spacer regions of the sRNA. We show that the ALFR motif and the spacer sequence adjacent to box C or C' are important for box C/D sRNP assembly in vitro. These data therefore reveal new RNA-protein contacts in the box C/D sRNP and suggest a role for Nop5 in substrate binding and/or release.

The enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction.

The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus--a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins.

Minor spliceosome components are predominantly localized in the nucleus.

Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.