Imp3 unfolds stem structures in pre-rRNA and U3 snoRNA to form a duplex essential for small subunit processing

Eukaryotic ribosome biogenesis requires rapid hybridization between the U3 snoRNA and the pre-rRNA to direct cleavages at the A₀, A₁, and A₂ sites in pre-rRNA that liberate the small subunit precursor. The bases involved in hybridization of one of the three duplexes that U3 makes with pre-rRNA, designated the U3-18S duplex, are buried in conserved structures: box A/A′ stem–loop in U3 snoRNA and helix 1 (H1) in the 18S region of the pre-rRNA. These conserved structures must be unfolded to permit the necessary hybridization. Previously, we reported that Imp3 and Imp4 promote U3-18S hybridization in vitro, but the mechanism by which these proteins facilitate U3-18S duplex formation remained unclear. Here, we directly addressed this question by probing base accessibility with chemical modification and backbone accessibility with ribonuclease activity of U3 and pre-rRNA fragments that mimic the secondary structure observed in vivo. Our results demonstrate that U3-18S hybridization requires only Imp3. Binding to each RNA by Imp3 provides sufficient energy to unfold both the 18S H1 and the U3 box A/A′ stem structures. The Imp3 unfolding activity also increases accessibility at the U3-dependent A₀ and A₁ sites, perhaps signaling cleavage at these sites to generate the 5′ mature end of 18S. Imp4 destabilizes the U3-18S duplex to aid U3 release, thus differentiating the roles of these proteins. Protein-dependent unfolding of these structures may serve as a switch to block U3-pre-rRNA interactions until recruitment of Imp3, thereby preventing premature and inaccurate U3-dependent pre-rRNA cleavage and folding events in eukaryotic ribosome biogenesis.     

The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.     

Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.     

The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5'-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit.     

Exploration of pairing constraints identifies a 9 base-pair core within box C/D snoRNA-rRNA duplexes

2'-O-ribose methylation of eukaryotic ribosomal RNAs is guided by RNA duplexes consisting of rRNA and box C/D small nucleolar (sno)RNA sequences, the methylated sites invariably mapping five positions apart from the D box. Here we have analyzed the RNA duplex pairing constraints by investigating the features of 415 duplexes from the fungus, plant and animal kingdoms, and the evolution of those duplexes within the 124 sets they group into. The D-box upstream 1st and >or=15th positions consist of Watson-Crick base-pairs, G:U base-pairs and mismatched bases with ratios close to random assortments; these positions display single base differences in >60% of the RNA duplex sets. The D-box upstream 2nd to 11th positions have >90% Watson-Crick base-pairs; they display single base mutations with a U-shaped distribution of lower values of 0% and 1.6% at the methylated site 5th and 4th positions, and double compensatory mutations leading to new Watson-Crick base-pairs with an inverted U-shaped distribution of higher values at the 8th to 11th positions. Half of the single mutations at the 3rd to 11th positions resulted in G:U base-pairing, mainly through A-->G mutations in the rRNA strands and C-->T mutations in the snoRNA strands. Double compensatory mutations at the 3rd to 11th positions are extremely frequent, representing 36% of all mutations; they frequently arose from an A-->G mutation in the rRNA strands followed by a T-->C mutation in the snoRNA strands. Differences in the mutational pathways through which the rRNA and snoRNA strand evolved must be related to differences in the rRNA and snoRNA copy number and gene organization. Altogether these data identify the D-box upstream 3rd to 11th positions as box C/D snoRNA-rRNA duplex cores. The impact of the pairing constraints on the evolution of the 9 base-pair RNA duplex cores is discussed.     

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.     

Nucleolar proteins Bfr2 and Enp2 interact with DEAD-box RNA helicase Dbp4 in two different complexes

Different pre-ribosomal complexes are formed during ribosome biogenesis, and the composition of these complexes is highly dynamic. Dbp4, a conserved DEAD-box RNA helicase implicated in ribosome biogenesis, interacts with nucleolar proteins Bfr2 and Enp2. We show that, like Dbp4, Bfr2 and Enp2 are required for the early processing steps leading to the production of 18S ribosomal RNA. We also found that Bfr2 and Enp2 associate with the U3 small nucleolar RNA (snoRNA), the U3-specific protein Mpp10 and various pre-18S ribosomal RNA species. Thus, we propose that Bfr2, Dbp4 and Enp2 are components of the small subunit (SSU) processome, a large complex of ∼80S. Sucrose gradient sedimentation analyses indicated that Dbp4, Bfr2 and Enp2 sediment in a peak of ∼50S and in a peak of ∼80S. Bfr2, Dbp4 and Enp2 associate together in the 50S complex, which does not include the U3 snoRNA; however, they associate with U3 snoRNA in the 80S complex (SSU processome). Immunoprecipitation experiments revealed that U14 snoRNA associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the ‘50S’ complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome.     

Janus chaperones: Assistance of both RNA- and protein-folding by ribosomal proteins

Ribosomal proteins assist the assembly and increase the stability of ribosomal RNA, without requiring ATP for their action. Some ribosomal proteins are also known to have essential functions outside the ribosome, i.e. promiscuity of functions that appears to correlate with their structural disorder. Here we addressed if certain ribosomal proteins with RNA chaperone activity and with a significant level of disorder also have protein-chaperone activity in vitro. Four proteins of the large subunit of Escherichia coli ribosome, L15, L16, L18 and L19 have been tested in three chaperone assays, in which all of them exhibited potent chaperone activity, commensurable with that of heat shock protein 90 kDa. These observations highlight possible novel aspects of the promiscuous functions of ribosomal proteins outside of the ribosome.     

Components of an interdependent unit within the SSU processome regulate and mediate its activity

The SSU processome is required for production of the small ribosomal subunit RNA, the 18S rRNA. Specifically, the U3 small nucleolar RNA (snoRNA) component of the SSU processome is essential for the formation of the conserved central pseudoknot and for cleavages of the pre-rRNA, both of which are required for 18S maturation. To further elucidate how these events are mediated, we examined the regulatory and mechanistic roles of the U3 specific proteins: Imp3p, Imp4p, and Mpp10p. We found that these proteins demonstrated an interdependence with respect to their stability and to their association with the U3 snoRNA. Because mutations in the U3 snoRNA that disrupt pre-rRNA processing confer similar defects on growth and pre-rRNA processing as do carboxy-terminal truncations of Mpp10p, we hypothesized that Mpp10p may be involved in maintaining U3 snoRNA-pre-rRNA base pairing. However, combining the two mutations resulted in a more pronounced cleavage defect at site A(2), suggesting that Mpp10p is also required at an additional mechanistic step. Furthermore, heterologous complementation experiments demonstrate that the last 95 amino acids of yeast Mpp10p are specifically required for growth and pre-rRNA processing at low temperatures.     

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.     

Ribosome biogenesis requires a highly diverged XRN family 5′→3′ exoribonuclease for rRNA processing in Trypanosoma brucei

Although biogenesis of ribosomes is a crucial process in all organisms and is thus well conserved, Trypanosoma brucei ribosome biogenesis, of which maturation of rRNAs is an early step, has multiple points of divergence. Our aim was to determine whether in the processing of the pre-rRNA precursor molecule, 5′→3′ exoribonuclease activity in addition to endonucleolytic cleavage is necessary in T. brucei as in other organisms. Our approach initiated with the bioinformatic identification of a putative 5′→3′ exoribonuclease, XRNE, which is highly diverged from the XRN2/Rat1 enzyme responsible for rRNA processing in other organisms. Tagging this protein in vivo allowed us to classify XRNE as nucleolar by indirect immunofluorescence and identify by copurification interacting proteins, many of which were ribosomal proteins, ribosome biogenesis proteins, and/or RNA processing proteins. To determine whether XRNE plays a role in ribosome biogenesis in procyclic form cells, we inducibly depleted the protein by RNA interference. This resulted in the generation of aberrant preprocessed 18S rRNA and 5′ extended 5.8S rRNA, implicating XRNE in rRNA processing. Polysome profiles of XRNE-depleted cells demonstrated abnormal features including an increase in ribosome small subunit abundance, a decrease in large subunit abundance, and defects in polysome assembly. Furthermore, the 5′ extended 5.8S rRNA in XRNE-depleted cells was observed in the large subunit, monosomes, and polysomes in this gradient. Therefore, the function of XRNE in rRNA processing, presumably due to exonucleolytic activity very early in ribosome biogenesis, has consequences that persist throughout all biogenesis stages.     

The U18 snRNA is not essential for pre-rRNA processing in Xenopus laevis.

The U18 small nuclear RNA (snRNA) is one of several newly discovered intron-encoded nucleolar RNAs whose function is unknown. We have studied the accumulation and function of the U18 snRNA in oocytes of the vertebrate, Xenopus laevis. The U18 snRNA contains 13 nt complementary to a highly conserved sequence in 28S ribosomal RNA (rRNA). Three oligonucleotides, selected to contain all or some of the complementary sequence, deplete the U18 snRNA upon injection into Xenopus oocytes. Injection of two of the oligonucleotides has no effect on pre-rRNA processing or ribosome transport. Injection of the third oligonucleotide does interrupt pre-18S rRNA processing, but this is due to coincidental simultaneous depletion of the U22 snRNA. The U18 snRNA is the first nucleolar snRNA that is not essential for ribosome biogenesis in vertebrates.     

Nop9 is an RNA binding protein present in pre-40S ribosomes and required for 18S rRNA synthesis in yeast

Proteomic analyses in yeast have identified a large number of proteins that are associated with preribosomal particles. However, the product of the yeast ORF YJL010C, herein designated as Nop9, failed to be identified in any previous physical or genetic analysis of preribosomes. Here we report that Nop9 is a nucleolar protein, which is associated with 90S and 40S preribosomes. In cells depleted of Nop9p, early cleavages of the 35S pre-rRNA are inhibited, resulting in the nucleolar retention of accumulated precursors and a failure to synthesize 18S rRNA. Nop9 contains multiple pumilio-like putative RNA binding repeats and displays robust in vitro RNA binding activity. The identification of Nop9p as a novel, essential factor in the nuclear maturation of 90S and pre-40S ribosomal subunits shows that the complement of ribosome synthesis factors remains incomplete.     

The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes

Ribosome biogenesis requires a vast number of trans-acting factors many of which are required for the chemical modification and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60–80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.     

Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

YqeH, a circularly permuted GTPase, is conserved among bacteria and eukaryotes including humans. It was shown to be essential for the assembly of small ribosomal (30S) subunit in bacteria. However, whether YqeH interacts with 30S ribosome and how it may participate in 30S assembly are not known. Here, using co-sedimentation experiments, we report that YqeH co-associates with 30S ribosome in the GTP-bound form. In order to probe whether YqeH functions as RNA chaperone in 30S assembly, we assayed for strand dissociation and annealing activity. While YqeH does not exhibit these activities, it binds a non-specific single and double-stranded RNA, which unlike the 30S binding is independent of GTP/GDP binding and does not affect intrinsic GTP hydrolysis rates. Further, S5, a ribosomal protein which participates during the initial stages of 30S assembly, was found to promote GTP hydrolysis and RNA binding activities of YqeH.     

Crystal Structure of the Yeast Ribosomal Protein rpS3 in Complex with Its Chaperone Yar1

Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8 Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly.     

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N⁷-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.