The 3′ ends of animal replication-dependent histone mRNAs are formed by endonucleolytic cleavage of the primary transcripts downstream of a highly conserved RNA hairpin. The hairpin-binding protein (HBP) binds to this RNA element and is involved in histone RNA 3′ processing. A minimal RNA-binding domain (RBD) of ∼73 amino acids that has no similarity with other known RNA-binding motifs was identified in human HBP [Wang Z-F et al., Genes & Dev, 1996, 10:3028-3040]. The primary sequence identity between human and Caenorhabditis elegans RBDs is 55% compared to 38% for the full-length proteins. We analyzed whether differences between C. elegans and human HBP and hairpins are reflected in the specificity of RNA binding. The C. elegans HBP and its RBD recognize only their cognate RNA hairpins, whereas the human HBP or RBD can bind both the mammalian and the C. elegans hairpins. This selectivity of C. elegans HBP is mostly mediated by the first nucleotide in the loop, which is C in C. elegans and U in all other metazoans. By converting amino acids in the human RBD to the corresponding C. elegans residues at places where the latter deviates from the consensus, we could identify two amino acid segments that contribute to selectivity for the first nucleotide of the hairpin loop.

U16 belongs to the family of box C/D small nucleolar RNAs (snoRNAs) whose members participate in ribosome biogenesis, mainly acting as guides for site-specific methylation of the pre-rRNA. Like all the other members of the family, U16 is associated with a set of protein factors forming a ribonucleoprotein particle, localized in the nucleolus. So far, only a few box C/D-specific proteins are known: in Xenopus laevis, fibrillarin and p68 have been identified by UV crosslinking and shown to require the conserved boxes C and D for snoRNA interaction. In this study, we have identified an additional protein factor (p62), common to box C/D snoRNPs, that crosslinks to the internal stem region, distinct from the conserved box C/D “core motif,” of U16 snoRNA. We show here that, although the absence of the core motif and, as a consequence, of fibrillarin and p68 binding prevents processing and accumulation of the snoRNA, the lack of the internal stem does not interfere with the efficient release of U16 from its host intron and only slightly affects snoRNA stability. Because this region is likely to be the binding site for p62, we propose that this protein plays an accessory role in the formation of a mature and stable U16 snoRNP particle.

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 μM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.

Loop–loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop–loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5′ noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop–loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.

An iterative in vitro splicing strategy was employed to select for optimal 3′ splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3′ end of the randomized insert. Mutating the 3′ terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6–7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by crosslinking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5′-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions −3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position −3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.

Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5′ terminus of 16S rRNA, in the 300 region of its 5′ domain, and in the 1070 region of its 3′-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-Å map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein–protein and protein–RNA crosslinking, chemical and enzymatic footprinting, and genetics.

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA–mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3′ ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3′ ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3′ ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3′ ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3′ ends were not uridylated in vitro, or when U6 snRNA with 3′ AOH was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3′ end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3′ uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3′ end of some small RNAs and suggest that one of the functions of the 3′ adenylation may be to negatively affect the 3′ uridylation of small RNAs.

Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.

Subdomain IIId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IIId, to explore the structural features of this subdomain. The proposed secondary structure of IIId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IIId. We found that this fragment contains several noncanonical structural motifs and non-Watson–Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA α-sarcin/ricin loop was located in the internal loop. The terminal loop, 5′-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U˚G base pair. The sixth nucleotide was bulged out to allow stacking of this U˚G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

Human endogenous retrovirus K (HERV-K) is the name given to an ∼30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome. Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1. HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev. Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the ∼416-nt K-RRE. Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE. Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure. Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev. Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

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.

tRNA–guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TΨC arm of in vitro-transcribed yeast tRNAPhe. Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TΨC arm of the yeast tRNAPhe indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TΨC stem into the loop). Posttranscriptional base modifications in tRNAPhe block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNAAsp. Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3′ untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is ∼15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

Eukaryotic nucleoli contain a large family of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs) that are involved in processing and site-specific methylation of pre-rRNA. Several proteins have been reported to be common factors of box C/D snoRNPs in lower and higher eukaryotes; nevertheless none of them has been clearly shown to directly interact with RNA. We previously identified in Xenopus laevis, by means of UV crosslinking in vivo, two proteins associated with box C/D snoRNAs, fibrillarin and p68. Here we show that fibrillarin interacts directly and specifically with the U16 box C/D snoRNA in a X. laevis oocyte nuclear extract and that it does not require p68 for binding. Specific binding is also obtained with a recombinant fibrillarin demonstrating that the protein is able to bind directly and specifically to U16 snoRNA by itself.

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2′,3′ cyclic phosphate and 5′-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNAMet has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2′,3′ cyclic phosphate at the 3′ end of the 5′ exon is converted into the splice junction phosphate forming a 3′,5′-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between “accurate” and “error-prone” conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.