Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.     

Chaperoning ribosome assembly

Reconstitution of bacterial ribosomes in vitro from RNA and protein constituents requires a heating step to rearrange conformation of an intermediate. In this issue of Molecular Cell, Maki et al. demonstrate that the DnaK chaperone system circumvents the requirement for heating.     

The essential GTPase YqeH is required for proper ribosome assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.     

Powering through ribosome assembly

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.     

The Escherichia coli GTPase CgtAE is involved in late steps of large ribosome assembly

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.     

MPV17L2 is required for ribosome assembly in mitochondria

MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ⁰ cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.     

A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly.

Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.     

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A₀, A₁, and A₂. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.     

RNA folding and ribosome assembly

Ribosome synthesis is a tightly regulated process that is crucial for cell survival. Chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. Rapid folding of the rRNA provides a platform for protein-induced assembly of the bacterial 30S ribosome. Multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and control the quality of the mature subunits.     

The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli

RluD is the pseudouridine synthase responsible for the formation of Psi1911, Psi1915, and Psi1917 in Escherichia coli 23S rRNA. Previous work from our laboratory demonstrated that disruption of the rluD gene and/or loss of the pseudouridine residues for which it is responsible resulted in a severe growth phenotype. In the current work we have examined further the effect of the loss of the RluD protein and its product pseudouridine residues in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S and 39S particles. Analysis of the 39S particles indicates that they are immature precursors of the 50S subunits, whereas the 62S particles are derived from the breakdown of unstable 70S ribosomes. In addition, purified mutant 70S ribosomes were found to be somewhat less efficient than wild type in protein synthesis. The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of Psi1911, Psi1915, and Psi1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of pseudouridine formation. The data suggest that the loss of the pseudouridine residues can account for all aspects of the mutant phenotype; however, a possible second function of the RluD synthase is also discussed.     

Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.

Summary We have examined mitochondrial (mt) ribosome assembly and-function in five nuclear and six extranuclear mutants of Neurospora crassa which had previously been characterized as deficient in cytochromes b and aa ₃. All six extranuclear mutants showed phenotypes similar to that previously described for the extranuclear [poky] mutant: small subunit-deficient with 19 S rRNA rapidly degraded. The nuclear mutants have the following phenotypes: 297-24 is mt small subunit deficient with 19 S RNA rapidly degraded. 289-56 is mt small subunit deficient but contains normal ratios of 19 S to 25 S RNA in whole mitochondria. 289-67 and 299-9 show defects in the processing of 25 S RNA leading to accumulation of a large precursor RNA. 289-4 is deficient in large subunits although a substantial, but less than normal, amount of 25 S RNA is present in the mitochondria.The present work provides new insight into the phenotypes of mt small subunit-deficient mutants. Previous studies using chloramphenicol suggest that some defects in the assembly of mt small subunits may arise secondarily as a result of inhibition of mt protein synthesis (LaPolla and Lambowitz, 1977; Lambowitz et al., 1979). Three mutants (289-56, 289-67 and 299-9) appear to show such defects. These strains contain incomplete mt small subunits which sediment more slowly than normal and are deficient in at least two proteins, S-5 and S-9. Correlation of mutant phenotypes with rates of mt protein synthesis in the different strains suggests that mt protein synthesis must be decreased to less than one half of the wild-type rate before secondary defects in mt small subunit assembly are observed. This threshold value is much lower than that which leads to gross deficiencies of cytochromes b and aa ₃. Although several mutants have phenotypes suggestive of alterations in mt ribosomal proteins, no such alterations could be identified by two dimensional gel electrophoresis.     

Biophysical studies of bacterial ribosome assembly

The assembly of the bacterial ribosome involves the association of over 50 proteins to 3 large RNA molecules, and it represents a major metabolic activity for rapidly growing bacteria. The availability of atomic structures of the ribosome and the application of biochemical and biophysical methods have led to rapid progress in understanding the mechanistic details of ribosome assembly. The basic steps required to assemble a ribosome are outlined, and the contributions of mass spectrometry, computational methods, and RNA-folding studies in understanding these steps are detailed. This complex process takes place with both sequential and parallel processing that is coordinated to ensure efficient and complete assembly of ribosomes to meet the demands of cell growth.     

The small subunit processome is required for cell cycle progression at G1

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.     

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.     

Abnormal ribosome assembly in a mutant of Escherichia coli.

The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown [Markey, Sims & Wild (1976) Biochem. J. 158, 451--456] to be an unusual intermediate in the assembly of 50S ribosomal subunits...     

The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis

In this paper the essential GTPase YlqF is shown to participate in the biogenesis of the 50S ribosomal subunit in Bacillus subtilis. Cells depleted of YlqF displayed gene expression profiles and nucleoid morphologies that were consistent with a function for YlqF in translation. In addition, YlqF is evolutionarily linked to two eukaryotic GTPases, Nog2p and Nug1p, that are involved in the biogenesis and the nuclear export of the 60S ribosomal subunit. Analysis of ribosomes from cells depleted of YlqF demonstrated that the formation of 70S ribosomes was greatly reduced and the large subunit sedimented at 45S. Cells grown with varying depleted levels of YlqF, yielding doubling times ranging from 38 min to 150 min, all displayed the 45S intermediate. Purified YlqF-His(6) protein associates with the 45S intermediate, but not the mature 50S subunit in vitro. Analysis of proteins from the 45S intermediate indicated that ribosomal protein L16, which is added late during in vitro Escherichia coli 50S ribosome biogenesis, was missing from the 45S intermediate. These results support a model in which YlqF participates in the formation of active 70S ribosomes in the cell by functioning in a late step of 50S subunit biogenesis. Based on these results we propose to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).     

The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lens- specific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek phi alpha kappa omicron sigma = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3% identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it therefore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phakinin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens fiber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximately 3:1. Under in vitro conditions, phakinin self- assembles into metastable filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 yields stable 10-nm filaments which have a smooth surface and are ultrastructurally indistinguishable from "mainstream" IFs. Immunolabeling with specific antibodies shows that these filaments represent phakinin/filensin heteropolymers. Despite its homology to the cytokeratins, phakinin does not coassemble with acidic (type I), or basic (type II) cytokeratins. From these data we conclude that filensin and phakinin are obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.