U3 Small Nucleolar RNA Research Articles

Abstract 6985: The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck cancer

Abstract Head and neck cancer (HNC) is the sixth common malignancy in the world and > 90% of HNC cases are squamous cell carcinoma in origin. Oral cancer is one major HNC subtype. Despite the recent advancements in cancer diagnostics and therapeutics, the 5-yr survival rate of advanced HNC patients remains <50%. Ribosomes are the cellular factories responsible for making proteins. The hyper-activation of ribosome biogenesis initiated by oncogene gain or tumor suppressor gene loss plays a critical role in cancer initiation and progression. Ribosome biogenesis is a complex and energy intensive process that involves hundreds of factors. UTP14A gene encodes a member of the uridine triphosphate 14 family and is an essential component of a large ribonucleoprotein complex bound to the U3 small nucleolar RNA. Previous studies have demonstrated that UTP14A plays a key role in the synthesis of ribosomes and 18S rRNA, and is overexpressed in several malignancies. However, the biological functions and underlying mechanisms of UTP14A in HNC remain elusive. We found not only the increase of UTP14A mRNA expression in TCGA-HNC database but also its negative impact on patient clinical outcome. UTP14A depletion significantly impaired HNC cell proliferation, migration and invasion in vitro. Ectopic overexpression had the opposite effect, suggesting an oncogenic role for UTP14A in HNC. Through gene set enrichment analysis of pairwise RNA sequencing results, several genes involved in the process of epithelial mesenchymal transition (EMT) were potentially associated with manipulated UTP14A expression. We also detected a concordant change of lipid droplet numbers and its surface marker expression in UTP14A-manipulated cells, respectively, by BODIPY staining and immunhistochemical staining. Western blot analysis validated a positive relationship of EMT markers and COX-2, a potential mediator of lipid metabolism and EMT, with altered UTP14A expression. The involvement of COX2 in UTP14A mediation was confirmed by using both pharmacological inhibition and genetic manipulation. We also identified p38 as a central downstream effector but acting upstream from COX-2 in UTP14A mediation in oral cancer cells. We believe that our study of UTP14A-mediated oncogenic signaling axis can potentially be harnessed as a novel therapeutic target against HNC although more mechanistic studies may be needed. Citation Format: Li-Wha Wu, Chen-Ni Chang, Pulak K. Biswas, Sen-Tien Tsai. The oncogenic functions and action mechanism of UTP14A, a subunit required for 18S rRNA synthesis, in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6985.

ABT1 modifies SMARD1 pathology via interactions with IGHMBP2 and stimulation of ATPase and helicase activity.

SMA with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) are results of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is a UPF1-like helicase with proposed roles in several cellular processes, including translation. This study examines activator of basal transcription 1 (ABT1), a modifier of SMARD1-nmd disease pathology. Microscale thermophoresis and dynamic light scattering demonstrate that IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2/ABT1 complex interacts with the 47S pre-rRNA 5' external transcribed spacer and U3 small nucleolar RNA (snoRNA), suggesting that the IGHMBP2/ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan, and substantially decreases neuromuscular junction denervation. To our knowledge, ABT1 is the first disease-modifying gene identified for SMARD1. We provide a mechanism proposing that ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide insight into SMARD1 pathogenesis, suggesting that ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.

Modification of Adenosine196 by Mettl3 Methyltransferase in the 5'-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation.

Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.

Impaired chondrcoyte U3 SNORNA expression in osteoarthritis and its impact on the chondrocyte's protein translation apparatus

Purpose: Osteoarthritis (OA) is the most prevalent degenerative joint disease with no disease-modifying treatment available. During OA progression, articular chondrocyte homeostasis is disturbed and presents with a catabolic signature. Pathways controlling ribosome activity have previously been described in the regulation of chondrocyte homeostasis. However, ribosome biogenesis has not been implicated in OA. We hypothesized that U3 small nucleolar RNA (snoRNA), a key factor in the endoribonucleolytic ribosomal RNA (rRNA) maturation, is critical for chondrocyte homeostasis via regulating the availability of mature rRNAs.

DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis.

The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

The yeast C/D box snoRNA U14 adopts a “weak” K-turn like conformation recognized by the Snu13 core protein in solution

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3′ side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5′ side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2′-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U•U and G•A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.

The human RNA helicase DHX37 is required for release of the U3 snoRNP from pre-ribosomal particles

ABSTRACTRibosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.

Evolution of Fungal U3 snoRNAs: Structural Variation and Introns.

The U3 small nucleolar RNA (snoRNA) is an essential player in the initial steps of ribosomal RNA biogenesis which is ubiquitously present in Eukarya. It is exceptional among the small nucleolar RNAs in its size, the presence of multiple conserved sequence boxes, a highly conserved secondary structure core, its biogenesis as an independent gene transcribed by polymerase III, and its involvement in pre-rRNA cleavage rather than chemical modification. Fungal U3 snoRNAs share many features with their sisters from other eukaryotic kingdoms but differ from them in particular in their 5’ regions, which in fungi has a distinctive consensus structure and often harbours introns. Here we report on a comprehensive homology search and detailed analysis of the evolution of sequence and secondary structure features covering the entire kingdom Fungi.

Network of epistatic interactions within a yeast snoRNA.

Epistatic interactions play a fundamental role in molecular evolution, but little is known about the spatial distribution of these interactions within genes. To systematically survey a model landscape of intragenic epistasis, we quantified the fitness of ~60,000 Saccharomyces cerevisiae strains expressing randomly mutated variants of the 333-nucleotide-long U3 small nucleolar RNA (snoRNA). The fitness effects of individual mutations were correlated with evolutionary conservation and structural stability. Many mutations had small individual effects but had large effects in the context of additional mutations, which indicated negative epistasis. Clusters of negative interactions were explained by local thermodynamic threshold effects, whereas positive interactions were enriched among large-effect sites and between base-paired nucleotides. We conclude that high-throughput mapping of intragenic epistasis can identify key structural and functional features of macromolecules.

DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

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.

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.

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Transcriptional repressor NIR functions in the ribosome RNA processing of both 40S and 60S subunits.

BackgroundNIR was identified as an inhibitor of histone acetyltransferase and it represses transcriptional activation of p53. NIR is predominantly localized in the nucleolus and known as Noc2p, which is involved in the maturation of the 60S ribosomal subunit. However, how NIR functions in the nucleolus remains undetermined. In the nucleolus, a 47S ribosomal RNA precursor (pre-rRNA) is transcribed and processed to produce 18S, 5.8S and 28S rRNAs. The 18S rRNA is incorporated into the 40S ribosomal subunit, whereas the 28S and 5.8S rRNAs are incorporated into the 60S subunit. U3 small nucleolar RNA (snoRNA) directs 18S rRNA processing and U8 snoRNA mediates processing of 28S and 5.8 S rRNAs. Functional disruption of nucleolus often causes p53 activation to inhibit cell proliferation.Methodology/Principal FindingsWestern blotting showed that NIR is ubiquitously expressed in different human cell lines. Knock-down of NIR by siRNA led to inhibition of the 18S, 28S and 5.8S rRNAs evaluated by pulse-chase experiment. Pre-rRNA particles (pre-rRNPs) were fractionated from the nucleus by sucrose gradient centrifugation and analysis of the pre-RNPs components showed that NIR existed in the pre-RNPs of both the 60S and 40S subunits and co-fractionated with 32S and 12S pre-rRNAs in the 60S pre-rRNP. Protein-RNA binding experiments demonstrated that NIR is associated with the 32S pre-rRNA and U8 snoRNA. In addition, NIR bound U3 snoRNA. It is a novel finding that depletion of NIR did not affect p53 protein level but de-repressed acetylation of p53 and activated p21.ConclusionsWe provide the first evidence for a transcriptional repressor to function in the rRNA biogenesis of both the 40S and 60S subunits. Our findings also suggested that a nucleolar protein may alternatively signal to p53 by affecting the p53 modification rather than affecting p53 protein level.

Does expression of the retrogene UTP14c in the ovary pre-dispose women to ovarian cancer?

It has been previously shown that the spermatogenesis associated retrogene, UTP14c, is expressed in over 50% of normal human ovaries and 80% of ovarian cancers. UTP14c is located on chromosome 13 as an intronless copy of the X-linked housekeeping gene, UTP14a. Like all spermatogenesis associated retrogenes, UTP14c is expressed in the testis and is essential for sperm production. It has no known role in the female and is not normally expressed in any cells or organs outside of the gonads. By comparison the protein encoded by UTP14a is found in all cell types and has a dual function. It is primarily involved in the biosynthesis of 18S ribosomal RNA in the nucleolus where it is a component of the U3 small nucleolar RNA associated protein complex. In addition, it down regulates TP53 in both the nucleus and cytoplasm by targeting it for proteolytic degradation. By analogy, we propose that the UTP14c peptide also targets TP53 for degradation. This in turn may prevent cells expressing UTP14c from entering apoptosis. The loss of TP53 in ovarian cells can also result in the down regulation of microRNA-145 (miR-145) expression. The loss of miR-145 can result in the activation of factors that promote oncogenesis and cellular pluripotency which in turn could lead to the development of ovarian cancer. We hypothesize that women, whose ovaries express UTP14c, are predisposed to ovarian cancer due to the disruption of protective signals that normally trigger TP53-mediated apoptosis and the dysregulation of genes that promote oncogenesis, such as c-Myc, that occurs when miR-145 synthesis is disrupted.

A second base pair interaction between U3 small nucleolar RNA and the 5′-ETS region is required for early cleavage of the yeast pre-ribosomal RNA

In eukaryotes, U3 snoRNA is essential for pre-rRNA maturation. Its 5′-domain was found to form base pair interactions with the 18S and 5′-ETS parts of the pre-rRNA. In Xenopus laevis, two segments of U3 snoRNA form base-pair interactions with the 5′-ETS region and only one of them is essential to the maturation process. In Saccharomyces cerevisiae, two similar U3 snoRNA–5′ ETS interactions are possible; but, the functional importance of only one of them had been tested. Surprisingly, this interaction, which corresponds to the non-essential one in X. laevis, is essential for cell growth and pre-rRNA maturation in yeast. In parallel with [Dutca et al. (2011) The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39, 5164–5180], here we show, that the second possible 11-bp long interaction between the 5′ domain of S. cerevisiae U3 snoRNA and the pre-rRNA 5′-ETS region (helix VI) is also essential for pre-rRNA processing and cell growth. Compensatory mutations in one-half of helix VI fully restored cell growth. Only a partial restoration of growth was obtained upon extension of compensatory mutations to the entire helix VI, suggesting sequence requirement for binding of specific proteins. Accordingly, we got strong evidences for a role of segment VI in the association of proteins Mpp10, Imp4 and Imp3.

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5' UTR of different HCV genotypes have not been available for testing 5' UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5' UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5' UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.

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.

RNA Chaperones Stimulate Formation and Yield of the U3 snoRNA–Pre-rRNA Duplexes Needed for Eukaryotic Ribosome Biogenesis

Short duplexes between the U3 small nucleolar RNA and the precursor ribosomal RNA must form quickly and with high yield to satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells. These interactions, designated the U3–ETS (external transcribed spacer) and U3–18S duplexes, are essential to initiate the processing of small subunit ribosomal RNA. Previously, we showed that duplexes corresponding to those in Saccharomyces cerevisiae are only observed in vitro after addition of one of two proteins: Imp3p or Imp4p. Here, we used fluorescence-based and other in vitro assays to determine whether these proteins possess RNA chaperone activities and to assess whether these activities are sufficient to satisfy the duplex yield and rate requirements expected in vivo. Assembly of both proteins with the U3 small nucleolar RNA into a chaperone complex destabilizes a U3 stem structure, apparently to expose its 18S base-pairing site. As a result, the chaperone complex accelerates formation of the U3–18S duplex from an undetectable rate to one comparable with the intrinsic rate observed for hybridizing short duplexes. The chaperone complex also stabilizes the U3–ETS duplex by 2.7 kcal/mol. These chaperone activities provide high U3–ETS duplex yield and rapid U3–18S duplex formation over a broad concentration range to help ensure that the U3–precursor ribosomal RNA interactions limit neither ribosome biogenesis nor rapid cell growth. The thermodynamic and kinetic framework used is general and thus suitable for investigating the mechanism of action of other RNA chaperones.