U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis. Genetic evidence suggests they form an extensively base-paired structure within the spliceosome that is required for catalysis. Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome. To this end, we synthesized and purified large segments of human U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (Tm = 50–60 °C in the presence of 20 mM Mg2+) complex. To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked species were identified and characterized. The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 5′ to U2-U6 helix Ia and opposite the invariant AGC in U6. Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast. Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN. Using a synthetic RNA modeled after the 25S rRNA 3′ ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is reduced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD.

In higher plants, RNA–DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed.

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The “core” domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Replacing a cassette of 31 residues from Escherichia coli release factor 1 with the equivalent residues in release factor 2 gave a protein active in codon-specific binding to the ribosome but inactive in peptidyl-tRNA hydrolysis. Such a phenotype is also found unexpectedly with release factor 2 when expressed at high concentration in bacteria. Substituting threonine with the release factor 1 equivalent serine at position 246 within the cassette restored the impaired activity of the chimeric protein, and also that of inactive recombinant release factor 2, both in vitro and in vivo. The differences in activity are not due to posttranslational modifications or a lack of it at this residue. Random mutagenesis of codon 246 suggests that this position is pivotal for the function of the release factor, being able to affect differentially both its binding to the ribosome and its peptide release activities. We propose that amino acid 246 is close to a sharp turn (GGQ motif at position 250), and is essential for transmitting the signal from cognate codon recognition by correctly positioning the peptidyl-tRNA hydrolysis domain of the release factor into the peptidyltransferase center.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2′-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

The Suppressor of forked protein is the Drosophila homolog of the 77K subunit of human cleavage stimulation factor, a complex required for the first step of the mRNA 3′-end-processing reaction. We have shown previously that wild-type su(f) function is required for the accumulation of a truncated su(f) transcript polyadenylated in intron 4 of the gene. This led us to propose a model in which the Su(f) protein would negatively regulate its own accumulation by stimulating 3′-end formation of this truncated su(f) RNA. In this article, we demonstrate this model and show that su(f) autoregulation is tissue specific. The Su(f) protein accumulates at a high level in dividing tissues, but not in nondividing tissues. We show that this distribution of the Su(f) protein results from stimulation by Su(f) of the tissue-specific utilization of the su(f) intronic poly(A) site, leading to the accumulation of the truncated su(f) transcript in nondividing tissues. Utilization of this intronic poly(A) site is affected in a su(f) mutant and restored in the mutant with a transgene encoding wild-type Su(f) protein. These data provide an in vivo example of cell-type-specific regulation of a protein level by poly(A) site choice, and confirm the role of Su(f) in regulation of poly(A) site utilization.

Reverse transcription of HIV-1 viral RNA uses human tRNA3Lys as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA3Lys expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA3Lys subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry Ψs, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, Ψ27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme–substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme–substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-RP phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-SP phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3′ end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qβ replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3′-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNAIle(UAU) and tRNAGln(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNAAsp was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than ∼17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNAIle as compared to the level of importation of the mainly cytosol-localized tRNAGln. Furthermore, exchanging the D-arm between the tRNAIle and the tRNAGln resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

Expression of the putative replicase of potato leafroll virus (PLRV) is regulated by −1 ribosomal frameshifting in which a primary viral transcript has two overlapping open reading frames (ORFs). A region of 39 nt at the junction of the two ORFs is essential for frameshifting to occur. It has been shown to harbor two signals, one active on the level of the primary structure, termed the slippery sequence, and one component that forms a secondary or tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frameshifting region and analyzed individual mutants for their ability to promote −1 frameshifting in vitro. Detailed comparison of our results with analogous mutants in the frameshifting region of the evolutionarily related beet western yellow virus, for which a crystal structure is available, unequivocally argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect putative pseudoknot-specific tertiary-base interactions drastically affect frameshifting activity. In addition, a specific deletion mutant was identified that displayed PLRV wild-type frameshifting activity with only 22 nt available for pseudoknot formation.

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a sequence of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt −6 to −29 could be folded into a potential hairpin structure with a loop represented by nt −16, −17, and −18, and a bulge involving nt −23. By introducing mutations that disrupted base pairing and compensatory mutations that restored base pairing, it was shown that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bottom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp activity, but mutation of A-23 into U or G had little effect on sgp activity. Mutation of loop residues A-16 and A-17 affected sgp activity, whereas mutation of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BMV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairpin structure is required for sgp activity.

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1–5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5′ end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5′ end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5′ end of deacylated tRNAPhe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45° between the respective planes of the molecules. The terminal phosphates of 3′CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

Bacteriophage φ6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only φ6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for φ6, and the packaging signals (pac sites) have been mapped to the 5′ ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of φ6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

We investigated the catalytic efficiency and the specificity of the Bacillus subtilis RNase P holoenzyme reaction with substrates that contain a single strand, a hairpin loop, or a tRNA 3′ to the cleavage site. At a saturating ribozyme concentration, RNase P can cleave a single-stranded RNA at ∼0.6 min−1 at pH 7.8. Replacing the single-stranded RNA 3′ to the cleavage site by a hairpin loop or by the yeast tRNAPhe increases the cleavage rate by up to ∼600-fold and ∼3,200-fold, respectively. These results show that compared to a single-stranded RNA substrate, the cleavage rate for the holoenzyme reaction is primarily enhanced by an acceptor-stem-like helix. Substrate binding, ∼7–10 μM for a single-stranded RNA, improves by ∼1,000-fold upon the addition of the tRNA. The efficiency of the RNase P holoenzyme cleaving a single-stranded RNA is sufficiently high to consider autolytic processing of the RNase P RNA (denoted P RNA) transcript in the cell. The addition of the RNase P protein to a precursor form of the P RNA in vitro results in autolytic processing of the 5′ and the 3′ end of this precursor in a matter of minutes. Autolytic processing produces the reported 5′ end of the mature P RNA. The precise 3′ end generated by autolytic processing is different over the course of the reaction and the final product is 4 nt shorter than the reported 3′ end of the B. subtilis P RNA. The observed 3′ end in vitro is consistent with the property of the holoenzyme reaction with single-stranded RNA substrates. The discrepancy with the reported 3′ end may be due to other processing events in vivo or inaccurate determination of the mature 3′ end of the P RNA isolated from the cell. We propose that the mature B. subtilis P RNA is generated at least in part by autolytic processing upon the binding of the RNase P protein to the precursor P RNA.

The complex of ribosomal protein L1 with 23S rRNA from Escherichia coli is of great interest because of the unique structural and functional aspects of this ribonucleoprotein domain. We have minimized the binding site for protein L1 on the 23S rRNA to nt 2120–2129, 2159–2162, and 2167–2178. This RNA fragment consists of two helices as well as an interconnecting loop of unknown structure. RNA molecules corresponding to the minimized L1 binding site, in which G, A, U, or C were individually replaced by their deoxyribo- (dN) or α-thio- (rNαS) analogs have been synthesized by T7 transcription in vitro and analyzed for their ability to bind protein L1. It has been demonstrated that the substitution of rNαS at position 2122 or 2176 decreases the affinity of the RNA for the protein in the presence of magnesium five- to tenfold, whereas the same changes have little effect on binding in the presence of manganese. This suggests that Rp oxygens in the phosphates preceding positions 2122 and 2176 are coordinated with Mg2+ and may participate in L1-23S rRNA interaction via magnesium bridges. We have also shown that this interaction is impaired by the presence of dC at position 2122 coupled with the presence of deoxyribonucleotide(s) at other positions in the RNA. This study demonstrates that the ribose–phosphate backbone of the helix encompassing nt 2120–2124/2174–2178 is intimately involved in the interaction of protein L1 with the 23S rRNA. In particular, we suggest that this helix is positioned in the cleft between the two domains of protein L1.