Reverse transcription of the HIV-1 RNA genome is primed by the cellular tRNAlys3 molecule that anneals to a complementary sequence in the viral genome, the primer-binding site (PBS). Additional interactions between the tRNA primer and the viral RNA were proposed to play a role in reverse transcription. We recently identified an 8-nt element in the U5 region upstream of the PBS that is critical for initiation and processive elongation of reverse transcription. This motif was termed the primer activation signal (PAS), and is proposed to interact with the “antiPAS sequence” in the TΨC arm of tRNAlys3. In this study, we demonstrate that the efficiency of initiation of reverse transcription can be modulated by PAS mutations that strengthen or weaken the interaction with antiPAS. These results provide further evidence for a direct base-pairing interaction between the PAS in the viral RNA and the antiPAS in the tRNAlys3 molecule. A broad phylogenetic survey indicated that a PAS element is present in all retroviral RNA genomes, suggesting that the process of reverse transcription is regulated by a common mechanism in all retroviridae. It has proven very difficult to change the identity of the tRNA primer for HIV-1 reverse transcription by changing the PBS sequence. Using in vitro reverse transcription assays, we demonstrate that the identity of the priming tRNA species can be switched by simultaneous alteration of the PBS and PAS motifs to accommodate a new tRNA primer. These results indicate that the PAS–antiPAS interaction is important for both primer selection and efficient reverse transcription.

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422–433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3–4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5′ domain. This creates the binding site for the Alu RNA 3′ domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3′ domain folding back by 180° onto the 5′ domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5′-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3′-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5′- and 3′-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3′-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5′ end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.

The internal ribosome entry segment (IRES) of picornaviruses consists of ∼450 nt of 5′-untranslated region, terminating at the 3′ end with an ∼25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and finally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in encephalomyocarditis virus (EMCV) virtually all ribosomes initiate translation at this site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3′ end of the IRES (at nt 586 in PV type 1) is considered to be a silent entry site, with all ribosomes initiating translation at the next AUG downstream (nt 743). To investigate what determines this different behavior, chimeras were constructed with a crossover at the conserved UUUC motif: the body of the IRES, the sequences upstream of this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of the FMDV or PV IRESes was replaced by that of EMCV, there was a marked increase in the absolute and relative frequency of initiation at the upstream AUG, the Lab site of FMDV and 586AUG of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, initiation occurred with no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the context of the AUG at the 3′ end of the IRES may influence initiation frequency at this site, as was shown by improving the context of 586AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-competent manner is particularly efficient and accurate with the EMCV IRES.

In established methods for analyzing ribozyme kinetics, radiolabeled RNA substrates are primarily used. Each data point requires the cumbersome sampling, gel electrophoretic separation, and quantitation of reaction products, apart from the continuous loss of substrate by radioactive decay. We have used stable, double fluorescent end-labeled RNA substrates. Fluorescence of one fluorophore is quenched by intramolecular energy transfer (FRET). Upon substrate cleavage, both dyes become separated in two RNA products and fluorescence is restored. This can be followed in real time and ribozyme reactions can be analyzed under multiple (substrate excess) and under single (ribozyme excess) turnover conditions. A detailed comparison of unlabeled, single, and double fluorescent-labeled RNAs revealed moderate kinetic differences. Results with two systems, hammerhead ribozymes in I/II (small ribozyme, large substrate) and in I/III format (large ribozyme, small substrate), are reported.

In mammals, as in all organisms examined, mRNAs that prematurely terminate translation are abnormally reduced in abundance by a mechanism called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (for reviews, see Maquat, 1995, 1996; Ruiz-Echevarria & Peltz, 1996; Li & Wilkinson, 1998; Culbertson, 1999; Hentze & Kulozik, 1999; Hilleren & Parker, 1999). This mechanism is thought to have evolved to eliminate nonsense-containing RNAs that arise as a consequence of (1) mutations in germ-line or somatic DNA or (2) routine abnormalities in gene expression due to abnormalities in, for example, transcription initiation, splicing, and somatic rearrangements of the type that characterize the immunoglobulin and T-cell receptor genes. The elimination of nonsense-containing mRNAs protects cells from the potentially deleterious effects of the encoded truncated proteins, which can manifest new or dominant-negative functions (Kazazian et al., 1992; Pulak & Anderson, 1993; Hall & Thein, 1994; Cali & Anderson, 1998). In addition to eliminating abnormal transcripts, NMD also regulates the expression of certain mRNAs that are not abnormal. Examples in mammalian cells are provided by certain selenoprotein mRNAs that terminate translation at a UGA codon for the inefficiently incorporated amino acid selenocysteine (Sec; Moriarty et al., 1997, 1998). Other examples will undoubtedly resemble natural substrates found in other organisms such as the alternatively spliced mRNAs of Caenorhabditis elegans that retain an internal exon harboring an in-frame termination codon (Morrison et al., 1997), and transcripts of Saccharomyces cerevisiae that contain a small open reading frame upstream of the primary open reading frame (Leeds et al., 1992; Pierrat et al., 1993).

Histone RNA 3′ processing in vitro produces one or more 5′ cleavage products corresponding to the mature histone mRNA 3′ end, and a group of 3′ cleavage products whose 5′ ends are mostly located several nucleotides downstream of the mRNA 3′ end. The formation of these 3′ products is coupled to the formation of 5′ products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3′ products therefore are a true and general feature of the processing reaction. Identical 3′ products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3′ end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3′ but no 5′ fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5′ end, indicating that product formation occurs by 5′-3′ exonucleolytic degradation. This degradation of a model 3′ product by a 5′-3′ exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5′ and 3′ extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5′ border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5′ flanking sequences. At the 3′ end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3′ 14 nt of these 23 nt do not enhance translation. The 5′ and 3′ sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5′ nucleotides base-pair with the 3′ nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3′ nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.

Binding of divalent metal ions plays a key role in the structure and function of ribozymes and other RNAs. In turn, the energetics and kinetics of the specific binding process are dominated by the balance between the cost of dehydrating the aqueous ion and the energy gained from inner-sphere interactions with the macromolecule. In this work, we introduce the use of the pulsed EPR technique of 2H Electron Spin-Echo Envelope Modulation (ESEEM) to determine the hydration level of Mn2+ ions bound to nucleotides and nucleic acids. Mn2+ is an excellent structural and functional mimic for Mg2+, the most common divalent ion of physiological interest. Comparison of data in D2O and H2O, with aqueous Mn2+ as a reference standard, allows a robust and precise determination of the number of bound water molecules, and therefore the number of RNA-derived ligands. Examples of applications to the mononucleotide models MnGMP and MnATP, as well as to the paradigmatic RNA system tRNAPhe, are shown.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250–570 μM at 25 °C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68–74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A9 metal-binding site and illustrates the symmetrical nature of the tandem G[bull ]A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3′ splice site of α-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3′ splice sites or mutated 5′ splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another.

The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on α-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3′ splice site of the regulated intron. We now report that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5′ splice site is required for efficient splicing inhibition by SXL. Psoralen-mediated crosslinking experiments suggest that SXL binding to this uridine-rich sequence inhibits recognition of the 5′ splice site by U1 snRNP in HeLa nuclear extracts. We also show that SXL interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5′ splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 5′ splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL.

Base pairing between the 5′ end of U1 snRNA and the conserved 5′ splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA–RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA–protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5′ splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5′ splice site and the 5′ end of U1 snRNA, without which the U1 snRNP–pre-mRNA complex is less stable and has a somewhat altered conformation.

A series of HeLa cell lines which stably express β-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5′ splice sites and activate a common 3′ cryptic splice site upstream leading to aberrantly spliced β-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5′ splice sites affected sensitivity of both the 5′ and 3′ splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5′ splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

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.

The Pb2+ cleavage of a specific phosphodiester bond in yeast tRNAPhe is the classical model of metal-assisted RNA catalysis. In vitro selection experiments have identified a tRNAPhe variant, the leadzyme, that is very active in cleavage by Pb2+. We present here a three-dimensional modeling protocol that was used to propose a structure for this ribozyme, and is based on the computation of the intersection of conformational space of sequence variants and the use of chemical modification data. Sequence and secondary structure data were used in a first round of computer modeling that allowed identification of conformations compatible with all known leadzyme variants. Common conformations were then tested experimentally by evaluating the activity of analogues containing modified nucleotides in the catalytic core. These experiments led to a new structural hypothesis that was tested in a second round of computer modeling. The resulting proposal for the active conformation of the leadzyme is consistent with all known structural data. The final model suggests an in-line SN2 attack mechanism and predicts two Pb2+ binding sites. The protocol presented here is generally applicable in modeling RNAs whenever the catalytic or binding activity of structural analogues is known.

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.

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3′ of the anticodon (position 37) in seven tRNA species, two of which are tRNACGGPro and tRNAGGGPro. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3′ of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6 tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6 tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.