Eukaryote ribosomal translation is terminated when release factor eRF1, in a complex with eRF3, binds to one of the three stop codons. The tertiary structure and dimensions of eRF1 are similar to that of a tRNA, supporting the hypothesis that release factors may act as molecular mimics of tRNAs. To identify the yeast eRF1 stop codon recognition domain (analogous to a tRNA anticodon), a genetic screen was performed to select for mutants with disabled recognition of only one of the three stop codons. Nine out of ten mutations isolated map to conserved residues within the eRF1 N-terminal domain 1. A subset of these mutants, although wild-type for ribosome and eRF3 interaction, differ in their respective abilities to recognize each of the three stop codons, indicating codon-specific discrimination defects. Five of six of these stop codon-specific mutants define yeast domain 1 residues (I32, M48, V68, L123, and H129) that locate at three pockets on the eRF1 domain 1 molecular surface into which a stop codon can be modeled. The genetic screen results and the mutant phenotypes are therefore consistent with a role for domain 1 in stop codon recognition; the topology of this eRF1 domain, together with eRF1-stop codon complex modeling further supports the proposal that this domain may represent the site of stop codon binding itself.

It has been suggested that in vitro selection experiments can provide information not only on what might have occurred during the evolution of the RNA world, but can in fact yield insights into particular features of the RNA world. In particular, it has been suggested that the sequences of anti-amino acid aptamers can provide clues to the origin of the genetic code, and that there is a statistically significant association between motifs found in aptamers and codons. We argue that the suggested connections between modern motifs and ancient sequences are logically tenuous, and show that there is no statistically meaningful association between motifs found in aptamers and codons.

Altman and colleagues (this issue) call attention to the inability of current standardized enzyme nomenclature to distinguish between enzymatic activities that reside in nonhomologous macromolecules. This issue is highlighted by the fact that the pre-tRNA 5′-maturation activities of bacteria and plant chloroplasts present the first instance (of which I am aware) of two naturally occurring enzymes that cannot be evolutionarily related, but which catalyze an identical reaction. (In the classic example of convergent evolution between the trypsin family and subtilisin, the enzymes do not have an identical substrate specificity.) Altman and colleagues propose that a single trivial name be used only for members of a family of homologous macromolecules; in other words, that different trivial names be given to enzymes that catalyze the same precursor–product conversion but do so with different catalytic mechanisms, or which are not members of a single family of homologous macromolecules.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3′ splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3′ splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3′ terminal intron residue. In this context, mutation of the conserved 3′ intron terminal G to a C also results in the partial activation of a nearby cryptic 3′ splice site with U as the 3′ terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3′ splice site over the normal 3′ splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6–U57 mutants to influence the fidelity of both branch site and 3′ splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein–RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1–2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3′ region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein–protein interactions. It has been previously shown that, in yeast, communication mediated by RNA–RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5′ splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement.

The methylation of 2′-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2′-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon–anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2′-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1Ser, using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2′-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2′-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2′-O-methyl nucleosides are not found in these positions within natural tRNA species.

Chemically modified nucleotide analogs have gained widespread popularity for probing structure–function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 Å resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3′-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3′ → 5′ exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5′ → 3′ exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5′ end is located closely downstream of the previously mapped 3′ end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Transcriptional regulation of the T box family of aminoacyl–tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specific interaction with the cognate uncharged tRNA. The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a “specifier sequence” in the leader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator. Previous studies have indicated that although these interactions are necessary for antitermination, they are unlikely to be sufficient. In the current study, the effect of multiple mutations in tRNATyr on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations in helical regions of tRNATyr that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not. The long variable arm of tRNATyr could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure. These results indicate that the tRNA–leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.

The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469–1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 °C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.

In higher eukaryotes, the regulation of pre-mRNA processing is still poorly known. The accumulation of various mature mRNAs, which can be observed in the nuclei of mammalian cells, is suggestive of a regulatory role of transport. However, the significance of these nuclear mRNA is presently unknown. We have used a tetracycline-regulated promoter to investigate the dynamics of these pools of mRNAs upon arrest of transcription. We observed, for β-globin and LT-α genes, a slow disappearance of these mRNA from the nucleus, with an apparent half-life that is similar to their cytoplasmic half-life. In view of these dynamics, these mRNA cannot simply be mature mRNAs in transit to the cytoplasm. They could be mRNAs retained in the nucleus, provided that the regulation of mRNA stability is comparable in the nucleus and the cytoplasm. But, because of their limited stability, these nuclear mRNAs cannot constitute a significant stock for gene expression. Alternatively, they could reflect a bidirectional transport of mRNA, that is, to and from the cytoplasm, which would provide a direct explanation for the similarity in both compartments of their half-life and poly(A) tail shortening over time.

If the genetic code arose in an RNA world, present codon assignments may reflect primordial RNA–amino acid affinities. Whether aptamers selected from random pools to bind free amino acids do so using the cognate codons at their binding sites has been controversial. Here we defend and extend our previous analysis of arginine binding sites, and propose a model for the maintenance of codon–amino acid interactions through the evolution of amino acids from ribozyme cofactors into the building blocks of proteins.

RNA polymerase II is implicated in the RNA-templated RNA synthesis during replication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA template nor protein factor requirements for this process are well defined. We have developed an in vitro transcription system based on HeLa cell nuclear extract (NE), in which a segment of antigenomic RNA corresponding to the left-hand tip region of the HDV rod-like structure serves as a template for efficient and highly specific RNA synthesis. Accumulation of the unique RNA product is highly sensitive to α-amanitin in HeLa NE and only partially sensitive to this drug in NE from PMG cells that contain an allele of the α-amanitin-resistant subunit of pol II, strongly suggesting pol II involvement in this reaction. Detailed analysis of the RNA product revealed that it represents a chimeric molecule composed of a newly synthesized transcript covalently attached to the 5′ half of the RNA template. Selection of the start site for transcription is remarkably specific and depends on the secondary structure of the RNA template, rather than on its primary sequence. Some features of this reaction resemble the RNA cleavage-extension process observed for pol II-arrested complexes in vitro. A possible involvement of the described reaction in HDV replication is discussed.

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing systems. We previously showed that 3′-sulfur substitution at the 3′ splice site of a nuclear pre-mRNA has no effect on splicing. We now report that 3′-sulfur substitution at the 3′ splice site of a nuclear pre-mRNA causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome uses a catalytic metal ion to stabilize the 3′-oxyanion leaving group during the second step of splicing, as shown previously for the first step. The lack of a metal-specificity switch under cis splicing conditions indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(−1) upstream of the 5′ splice site. P(−1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(−1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min−1 for the wild-type RNA to ∼4 min−1 at 30 °C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.