Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles

Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.     

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.     

A direct interaction between the Utp6 half-a-tetratricopeptide repeat domain and a specific peptide in Utp21 is essential for efficient pre-rRNA processing

The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 μM.     

Direct interaction between Utp8p and Utp9p contributes to rRNA processing in budding yeast

The small subunit (SSU) processome is an evolutionarily conserved ribonucleoprotein (RNP) complex that consists of U3 snoRNA and at least 40 protein components. The SSU processome is required for the generation of 18S rRNA in the budding yeast Saccharomyces cerevisiae. In this study we demonstrate that two essential components of the SSU processome, Utp8p and Utp9p, must interact directly for the SSU processome to function properly. Disruption of the Utp8p-Utp9p interaction by mutation of the respective interacting domain led to a compromised ability of yeast cells to process 35S pre-rRNA into 18S pre-rRNA. Loss of the Utp8p-Utp9p interaction also led to a decrease in the amount of Utp8p that interacted with U3 small nucleolar RNAs (snoRNAs) but did not affect the amount of Utp9p bound to U3 snoRNA, suggesting that Utp8p associates with the SSU processome by virtue of its interaction with Utp9p. Together, our data support a model where Utp8p and Utp9p must interact directly and functionally in the U3-containing SSU processome for optimal rRNA biosynthesis to occur in budding yeast.     

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.     

Structural and functional analysis of Utp23, a yeast ribosome synthesis factor with degenerate PIN domain

During synthesis of yeast ribosome, a large complex, called the 90S pre-ribosome or the small subunit processome, is assembled on the nascent precursor rRNA and mediates early processing of 18S rRNA. The Utp23 protein and snR30 H/ACA snoRNA are two conserved components of 90S pre-ribosomes. Utp23 contains a degenerate PIN nuclease domain followed by a long C-terminal tail and associates specifically with snR30. Here, we report the crystal structure of the Utp23 PIN domain at 2.5-Å resolution. The structure reveals a conserved core fold of PIN domain with degenerate active site residues, a unique CCHC Zn-finger motif, and two terminal extension elements. Functional sites of Utp23 have been examined with conservation analysis, mutagenesis, and in vivo and in vitro assays. Mutations in each of three cysteine ligands of zinc, although not the histidine ligand, were lethal or strongly inhibitory to yeast growth, indicating that the Zn-finger motif is required for Utp23 structure or function. The N-terminal helix extension harbors many highly conserved basic residues that mostly are critical for growth and in vitro RNA-binding activity of Utp23. Deletion of the C-terminal tail, which contains a short functionally important sequence motif, disrupted the interaction of Utp23 with snR30 and perturbed the pre-ribosomal association of Utp23. Our data establish a structural framework for dissecting Utp23 function in the assembly and dynamics of 90S pre-ribosomes.     

Ebp2p, the yeast homolog of Epstein-Barr virus nuclear antigen 1-binding protein 2, interacts with factors of both the 60 S and the 40 s ribosomal subunit assembly

Ebp2p, the yeast homolog of human Epstein-Barr virus nuclear antigen 1-binding protein 2, is essential for biogenesis of the 60 S ribosomal subunit. Two-hybrid screening exhibited that, in addition to factors necessary for assembly of the 60 S subunit, Ebp2p interacts with Rps16p, ribosomal protein S16, and the 40 S ribosomal subunit assembly factor, Utp11p, as well as Yil019w, the function of which was previously uncharacterized. Depletion of Yil019w resulted in reduction in levels of both of 18 S rRNA and 40 S ribosomal subunit without affecting levels of 25 S rRNA and 60 S ribosomal subunits. 35 S pre-rRNA and aberrant 23 S RNA accumulated, indicating that pre-rRNA processing at sites A(0)-A(2) is inhibited when Yil019w is depleted. Each combination from Yil019w, Utp11p, and Rps16p showed two-hybrid interaction.     

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.     

Utp14 Recruits and Activates the RNA Helicase Dhr1 To Undock U3 snoRNA from the Preribosome

In eukaryotic ribosome biogenesis, U3 snoRNA base pairs with the pre-rRNA to promote its processing. However, U3 must be removed to allow folding of the central pseudoknot, a key feature of the small subunit. Previously, we showed that the DEAH/RHA RNA helicase Dhr1 dislodges U3 from the pre-rRNA. DHR1 can be linked to UTP14, encoding an essential protein of the preribosome, through genetic interactions with the rRNA methyltransferase Bud23. Here, we report that Utp14 regulates Dhr1. Mutations within a discrete region of Utp14 reduced interaction with Dhr1 that correlated with reduced function of Utp14. These mutants accumulated Dhr1 and U3 in a pre-40S particle, mimicking a helicase-inactive Dhr1 mutant. This similarity in the phenotypes led us to propose that Utp14 activates Dhr1. Indeed, Utp14 formed a complex with Dhr1 and stimulated its unwinding activity in vitro. Moreover, the utp14 mutants that mimicked a catalytically inactive dhr1 mutant in vivo showed reduced stimulation of unwinding activity in vitro. Dhr1 binding to the preribosome was substantially reduced only when both Utp14 and Bud23 were depleted. Thus, Utp14 is bifunctional; together with Bud23, it is needed for stable interaction of Dhr1 with the preribosome, and Utp14 activates Dhr1 to dislodge U3.     

Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle

Here we report the functional characterization of Pwp2, an evolutionary conserved component of the 90 S pre-ribosome. Conditional depletion of the Pwp2 protein in yeast specifically impairs pre-rRNA proccessing at sites A(0), A(1), and A(2), leading to a strong decrease in 18 S rRNA and 40 S ribosomal subunit levels. Pre-ribosomal particle sedimentation analysis indicated that these defects are caused by a block in the formation of 90 S pre-ribosomes. We demonstrate that in Pwp2-depleted cells the U3 small nucleolar ribonucleoprotein is not able to interact with the 35 S pre-rRNA and accumulates as a free complex. Similarly, other 90 S particle components such as Imp3 and Imp4 do not associate with the pre-rRNA precursor in the absence of Pwp2. In addition, we have found that after blocking U3 ribonucleoprotein assembly, Pwp2 predominantly accumulates as a complex in association with five proteins: Dip2, Utp6, Utp13, Utp18, and Utp21. Immunoprecipitation and gradient sedimentation analysis revealed that this Pwp2 small subcomplex is capable of interacting directly with the 35 S pre-rRNA 5' end. Taken together, these results indicate that Pwp2 forms part of a stable particle subunit independent of the U3 small nucleolar ribonucleoprotein that is essential for the initial assembly steps of the 90 S pre-ribosome.     

Structure of Utp21 Tandem WD Domain Provides Insight into the Organization of the UTPB Complex Involved in Ribosome Synthesis

Assembly of the eukaryotic ribosome requires a large number of trans-acting proteins and small nucleolar RNAs that transiently associate with the precursor rRNA to facilitate its modification, processing and binding with ribosomal proteins. UTPB is a large evolutionarily conserved complex in the 90S small subunit processome that mediates early processing of 18S rRNA. UTPB consists of six proteins Utp1/Pwp1, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 and has abundant WD domains. Here, we determined the crystal structure of the tandem WD domain of yeast Utp21 at 2.1 Å resolution, revealing two open-clamshell-shaped β-propellers. The bottom faces of both WD domains harbor several conserved patches that potentially function as molecular binding sites. We show that residues 100–190 of Utp18 bind to the tandem WD domain of Utp21. Structural mapping of previous crosslinking data shows that the WD domains of Utp18 and Utp1 are organized on two opposite sides of the Utp21 WD domains. This study reports the first structure of a UTPB component and provides insight into the structural organization of the UTPB complex.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis

Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.     

The small-subunit processome is a ribosome assembly intermediate

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.     

An RNA-Binding Complex Involved in Ribosome Biogenesis Contains a Protein with Homology to tRNA CCA-Adding Enzyme

A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA–protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.     

Rok1p is a putative RNA helicase required for rRNA processing.

The synthesis of ribosomes involves many small nucleolar ribonucleoprotein particles (snoRNPs) as transacting factors. Yeast strains lacking the snoRNA, snR10, are viable but are impaired in growth and delayed in the early pre-rRNA cleavages at sites A0, A1, and A2, which lead to the synthesis of 18S rRNA. The same cleavages are inhibited by genetic depletion of the essential snoRNP protein Gar1p. Screens for mutations showing synthetic lethality with deletion of the SNR10 gene or with a temperature-sensitive gar1 allele both identified the ROK1 gene, encoding a putative, ATP-dependent RNA helicase of the DEAD-box family. The ROK1 gene is essential for viability, and depletion of Rok1p inhibits pre-rRNA processing at sites A0, A1, and A2, thereby blocking 18S rRNA synthesis. Indirect immunofluorescence by using a ProtA-Rok1p construct shows the protein to be predominantly nucleolar. These results suggest that Rok1p is required for the function of the snoRNP complex carrying out the early pre-rRNA cleavage reactions.     

Human 1A6/DRIM, the homolog of yeast Utp20, functions in the 18S rRNA processing

1A6/DRIM is a nucleolar protein with a nucleolar targeting sequence in its 3'-terminus. Bioinformatic analysis indicated that human 1A6/DRIM shares 23% identity and 43% similarity with yeast Utp20, which has been reported as a component of U3 snoRNA protein complex and has been implicated in 18S rRNA processing. In the present study, we found, by utilizing RT-PCR with RNA extracted from anti-1A6/DRIM immunoprecipitates and Northern blotting, that 1A6/DRIM is associated with U3 snoRNA. Pulse-chase labeling assays showed that silencing of 1A6/DRIM expression in HeLa cells resulted in a delayed 18S rRNA processing. Furthermore, immunoprecipitations revealed that 1A6/DRIM was also associated with fibrillarin, another U3 RNP component in HeLa cells. These results indicate that 1A6/DRIM is involved in 18S rRNA processing and is the bona fide mammalian Utp20.     

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.