By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5′ domain of BC1 RNA, derived from tRNAAla, does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNAAla domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3′ end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression. Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus. Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rpl25p, consistent with a role in subunit assembly. Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5.8SL and 5.8SS. In cells depleted for Nop7p, the 27SA3 pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8SS rRNA were depleted. Less depletion was seen for the 5.8SL pathway. TAP-tagged Nop7p coprecipitated precursors to both 5.8SL and 5.8SS but not the mature rRNAs. We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA3 pre-rRNA and has additional functions in 60S subunit assembly and transport. Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5′ viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5′ end of the CAT sequence, or the insertion of a small stable hairpin structure (ΔG = −18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5′ proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.

The genome of the positive strand RNA bacteriophage Qβ folds into a number of structural domains, defined by long-distance interactions. The RNA within each domain is ordered in arrays of three- and four-way junctions that confer rigidity to the chain. One such domain, RD2, is about 1,000-nt long and covers most of the replicase gene. Its downstream border is the 3′ untranslated region, whereas upstream the major binding site for Qβ replicase, the M-site, is located. Replication of Qβ RNA has always been puzzling because the binding site for the enzyme lies some 1,500-nt away from the 3′ terminus. We present evidence that the long-range interaction defining RD2 exists and positions the 3′ terminus in the vicinity of the replicase binding site. The model is based on several observations. First, mutations destabilizing the long-range interaction are virtually lethal to the phage, whereas base pair substitutions have little effect. Secondly, in vitro analysis shows that destabilizing the long-range pairing abolishes replication of the plus strand. Thirdly, passaging of nearly inactive mutant phages results in the selection of second-site suppressor mutations that restore both long-range base pairing and replication. The data are interpreted to mean that the 3D organization of this part of Qβ RNA is essential to its replication. We propose that, when replicase is bound to the internal recognition site, the 3′ terminus of the template is juxtaposed to the enzyme's active site.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea. In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more. We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNAPhe as a substrate. Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c. We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNATyr and pre-tRNALeu. Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide. Dus1 modifies yeast pre-tRNAPhe in vitro at U17, one of the two positions that are known to bear this modification in vivo. Yeast extract from a dus1-Δ strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-Δ and dus2-Δ strains is significantly depleted in dihydrouridine content.

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-Å-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined “barrel only” images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA. Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels. Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction. NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar. Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure. The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (kon) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5′ splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

The 3′ ends of most eukaryotic pre-mRNAs are generated by 3′ endonucleolytic cleavage and subsequent polyadenylation. 3′-end formation can be influenced positively or negatively by various factors. In particular, U1 snRNP acts as an inhibitor when bound to a 5′ splice site located either upstream of the 3′-end formation signals of bovine papilloma virus (BPV) late transcripts or downstream of the 3′-end processing signals in the 5′ LTR of the HIV-1 provirus. Previous work showed that in BPV it is not the first step, 3′ cleavage, that is affected by U1 snRNP, but rather the second step, polyadenylation, that is inhibited. Since in HIV-1 the biological requirement is to produce transcripts that read through the 5′ LTR cleavage site rather than being cleaved there, this mechanism seemed unlikely to apply. The obvious difference between the two examples was the relative orientation of the 3′-end formation signals and the U1 snRNP-binding site. In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3′ cleavage reaction. U1 snRNP was found to inhibit cleavage when bound to a 5′ splice site downstream of the cleavage/polyadenylation site, as in the HIV-1 LTR. U1 snRNP binding at this location was shown not to affect the recruitment of multiple cleavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP on polyadenylation of other pre-mRNAs, were shown not to be required for cleavage inhibition. Therefore, U1 snRNP bound to a 5′ splice site can inhibit cleavage and polyadenylation in two mechanistically different ways depending on whether the 5′ splice site is located upstream or downstream of the cleavage site.

The C-to-U editing of apolipoprotein-B (apo-B) mRNA is catalyzed by an enzyme complex that recognizes an 11-nt mooring sequence downstream of the editing site. A minimal holoenzyme that edits apo-B mRNA in vitro has been defined. This complex contains apobec-1, the catalytic subunit, and apobec-1 complementation factor (ACF), the RNA-binding subunit that binds to the mooring sequence. Here, we show that ACF binds with high affinity to single-stranded but not double-stranded apo-B mRNA. ACF contains three nonidentical RNA recognition motifs (RRM) and a unique C-terminal auxiliary domain. In many multi-RRM proteins, the RRMs mediate RNA binding and an auxiliary domain functions in protein–protein interactions. Here we show that ACF does not fit this simple model. Based on deletion mutagenesis, the RRMs in ACF are necessary but not sufficient for binding to apo-B mRNA. Amino acids in the pre-RRM region are required for complementing activity and RNA binding, but not for interaction with apobec-1. The C-terminal 196 amino acids are not absolutely essential for function. However, further deletion of an RG-rich region from the auxiliary domain abolished complementing activity, RNA binding, and apobec-1 interaction. The auxiliary domain alone did not bind apobec-1. Although all three RRMs are required for complementing activity and apobec-1 interaction, the individual motifs contribute differently to RNA binding. Point mutations in RRM1 or RRM2 decreased the Kd for apo-B mRNA by two orders of magnitude whereas mutations in RRM3 reduced binding affinity 13-fold. The pairwise expression of RRM1 with RRM2 or RRM3 resulted in moderate affinity binding.

Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3′ untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 Å resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer β/α/β sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349–2352), the interdomain angle differs by 33° in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a “gooseneck” elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also complexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show that the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequence and structure that are necessary for protein recognition. We identified a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA revealed that binding by the 60-kDa Ro protein requires specific base pairs within the conserved helix, a singly bulged nucleotide that disrupts the helix, and a three-nucleotide bulge on the opposing strand. Chemical probing experiments using diethyl pyrocarbonate demonstrated that, in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs in the conserved helix by binding in the major groove of the RNA. Furthermore, experiments in which dimethyl sulfate was used to probe a naked and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.

Posttranscriptional modifications were mapped in helices 90–92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90–92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2′-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (Ψs). A total of 10 posttranscriptionally methylated nucleotides and 6 Ψs were detected in the five organisms. Eight of the methylated nucleotides and one Ψ have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

The transcript of the Saccharomyces cerevisiae gene, RPL30, is subject to regulated splicing and regulated translation, due to a structure that interacts with its own product, ribosomal protein L30. We have followed the fate of the regulated RPL30 transcripts in vivo. Initially, these transcripts abortively enter the splicing pathway, forming an unusually stable association with U1 snRNP. A large proportion of the unspliced molecules, however, are found in the cytoplasm. Most of these are still bound by L30, as only a small fraction are engaged in translation. Eventually, the unspliced RPL30 transcripts escape the grasp of L30, associate with ribosomes, and fall prey to nonsense mediated decay.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA–CopT pairing in vitro showed that the dominant product of antisense RNA–mRNA binding is not a full RNA duplex. We have studied here the structure of CopA–CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop–loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA–CopT RNA complex. This stabilizer helix cannot form in CopI–CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI–CopT) and the stable complex (CopA–CopT) are discussed.

We have adapted the yeast three-hybrid system to identify RNA ligands for an RNA-binding protein. In this assay system, a protein–RNA interaction is detected by the reconstitution of a transcriptional activator using two hybrid proteins and a hybrid RNA. The RNA molecule is tethered to the promoter of a reporter gene by binding to a hybrid protein consisting of the bacteriophage MS2 coat protein fused to the DNA-binding protein LexA; the RNA-binding domain to be analyzed is fused to the transcriptional activation domain of the yeast Gal4 protein; and the bifunctional RNA consists of binding sites for the coat protein and for the other RNA-binding domain. We built an RNA library such that short fragments of genomic DNA from yeast were transcribed in yeast together with binding sites for the coat protein. We screened this hybrid RNA library for RNAs that bound to the yeast Snp1 protein, a homolog of the human U1-70K protein. The screen yielded as the strongest positive the fragment of U1 RNA that contains loop I, which is known to bind to Snp1 in U1 snRNP. We also identified four other RNA ligands that produced weaker three-hybrid signals, suggesting lower affinities for Snp1 as compared to U1 RNA. In addition, this search also yielded a set of RNA sequences that can activate transcription on their own when bound to a promoter through a protein interaction.