This review is occasioned by the fact that the problem of translation, which has simmered on the biological sidelines for the last 40 years, is about to erupt center stage—thanks to the recent spectacular advances in ribosome structure. This most complex, beautiful, and fascinating of cellular mechanisms, the translation apparatus, is also the most important. Translation not only defines gene expression, but it is the sine qua non without which modern (protein-based) cells would not have come into existence. Yet from the start, the problem of translation has been misunderstood—a reflection of the molecular perspective that dominated Biology of the last century. In that the our conception of translation will play a significant role in creating the structure that is 21st century Biology, it is critical that our current (and fundamentally flawed) view of translation be understood for what it is and be reformulated to become an all-embracing perspective about which 21st century Biology can develop. Therefore, the present review is both a retrospective and a plea to biologists to establish a new evolutionary, RNA-World-centered concept of translation. What is needed is an evolutionarily oriented perspective that, first and foremost, focuses on the nature (and origin) of a primitive translation apparatus, the apparatus that transformed an ancient evolutionary era of nucleic acid life, the RNA World, into the world of modern cells.

Protein synthesis in ribosomes requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. Synthesis is also assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by their anticodons is known. The termination factors are thought to do this. In the large ribosomal RNA, we found two sites that, like tRNAs, contain the anticodon hairpin but with triplets complementary to stop codons. One site is hairpin 69 from domain IV; the other site is hairpin 89, domain V. By analogy, we call them termination tRNAs: Ter-tRNA1 and Ter-tRNA2, respectively, even though they transport no amino acids, and suggest that they directly pair to stop codons. The termination factors only aid in this recognition, making it specific and reliable. A strong argument in favor of our hypothesis comes from vertebrate mitochondria. They are known to acquire two new stop codons, AGA and AGG. In the standard code, these are two out of six arginine codons. We revealed that the corresponding anticodons, UCU and CCU, have evolved in Ter-tRNA1 of these mitochondria.

For two decades, up to the mid-1980s, study of the translation phase of gene expression was conducted by a relatively small group of protein biochemistry, cell physiology, and developmental biology labs. These workers labored against a widely held view that anything and everything exciting in the control of gene expression must occur at the level of transcription. Since that time, however, the use of molecular genetics has revealed an amazing array of mechanisms for regulating both overall protein synthesis and selective control of translation of individual mRNAs in response to physiological conditions. This led to a remarkable influx of workers into the field, severely challenging the catering facilities at the Cold Spring Harbor Laboratory during the biannual meetings on Translational Control, which were inaugurated in 1987. The first Cold Spring Harbor “Translational Control” monograph was published in 1996, and its excellent articles have provided a valuable resource to established workers and newcomers alike. However, it is a reflection of the rapid development of knowledge in this field that this volume now looks decidedly middle-aged. It was therefore decided to produce a new volume of articles for the year 2000. Although a few of the 36 chapters in the new monograph are necessary updates of subjects covered previously, by far the majority of the articles are by different authors and cover new directions, many of which were barely a gleam in the eye in 1996.

Trans-splicing requires that 5′ and 3′ splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3′ splice site recognition in vitro. Efficient recognition of the 3′ splice site is dependent upon both the sequence of the 3′ splice site itself and enhancer elements located in the 3′ exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.

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.

Picornavirus internal ribosome entry sites (IRESs) are ∼450 nt. RNA elements that direct internal initiation of translation, such that when placed between the two cistrons of a dicistronic construct, they drive independent translation of the downstream cistron. Consequently they have been widely used for coordinated expression of two or more proteins. All picornavirus IRESs have an AUG triplet at the very 3′ end, which is thought to be the actual site of internal ribosome entry. However with some IRESs, such as foot-and-mouth disease virus, and especially poliovirus, the majority of ribosomes do not initiate translation at this putative entry site AUG, but at the next AUG further downstream, which is thought to be accessed by a process of linear ribosome scanning from the entry site. If this is so, then it should be possible to regulate IRES-dependent translation by inserting an iron responsive element (IRE) between the putative entry site AUG and the main functional initiation site. This should make IRES-dependent translation sensitive to the concentration of iron regulatory protein (IRP), the protein that specifically binds to the IRE. This has been attempted with both the foot-and-mouth disease virus and poliovirus IRESs, and was successful in so far as an inhibition specifically of IRES-dependent translation was observed that was strictly dependent on both the presence of IRP and of a functional IRE motif inserted in the sense orientation. However, the range over which expression could be varied was rather limited (three- to fourfold maximum), because some IRES-dependent translation remained completely refractory to inhibition by even very high IRP concentrations. In contrast, with a cap-proximal IRE in the 5′ untranslated region of an mRNA translated by the scanning mechanism, addition of sufficient IRP results in complete inhibition. These results support the model of IRES-promoted ribosome entry at an upstream site followed by strictly linear scanning to the main functional initiation site for the majority of internal initiation events, but imply that some ribosomes must access the functional initiation site by another route, possibly a nonlinear shunting-like mechanism.

The current nomenclature system of group I introns (see Cech, 1988; Michel & Westhof, 1990) has become insufficient to distinguish and categorize the complex collection of more than 900 group I introns in ribosomal DNA (rDNA) of nuclear, mitochondrial, chloroplast, and eubacterial genomes (http://www.rna.icmb.utexas.edu/; GenBank; our unpubl. results) in a rational way. The majority of these group I introns (∼750) are found in nuclear rDNA of fungi and protists, but the distribution appears highly scattered since most species analyzed lack introns. Many of the rDNA introns are optional among strains of a particular species or between closely related species, and some have been shown in experimental settings to be true mobile genetic elements (see Belfort & Roberts, 1997). All group I rDNA introns are found at a limited number of insertion sites (∼75) in highly conserved regions of the small subunit (SSU) and large subunit (LSU) rRNA genes, and some of these sites (∼10) are shared by introns from the nuclei, mitochondria, or chloroplasts. There are numerous examples of multiple group I introns in a single rRNA gene, and as many as eight nuclear introns have been noted in the SSU rDNA of the lichen ascomycete Lecanora dispersa (accession number L37734) and in the LSU rDNA of the myxomycete Fuligo septica (our unpubl. results). Finally, group I introns that occupy the same site in rDNA, but in distantly related hosts, tend to share a number of structural features as well as high levels of primary sequence similarities compared to introns at different insertion sites (e.g., Suh et al., 1999).

The use of double-stranded (ds) RNA to manipulate gene expression is evolving into an increasingly powerful tool to study gene function in organisms ranging from plants to invertebrates to mammals. Underlying the technological advances enabled by RNA interference (RNAi) is a rich biology. RNAi and related phenomena represent evolutionarily conserved mechanisms, which protect organisms from invasion by both exogenous (e.g., viruses) and endogenous (e.g., mobile genetic elements) genetic parasites. In addition, it is now clear that dsRNA-dependent silencing mechanisms represent a conserved regulatory motif for endogenous programs of gene expression. We are just beginning to understand not only the biological roles of RNAi but also the mechanistic basis of this process. The goal of this review is to familiarize the reader with the diversity of biological responses that may be related to RNA interference and to summarize the current state of knowledge regarding the mechanistic bases of these silencing phenomena.

A meeting “Ribosome Biogenesis and Nucleolar Function” was held at Lake Tahoe, California, August 17–21, 2000, ably organized by Lasse Lindahl and Janice Zengel (University of Maryland, Baltimore County), together with Maurille Fournier (University of Massachusetts), Craig Pikaard (Washington University), and John Woolford (Carnegie-Mellon University). The meeting was noteworthy in several respects. First, it occurred within only a few days (on either side) of the publication of breathtaking 2.4–3.3 Å structures of the large and small ribosomal subunits. Second, it addressed how this machine is built in the cell to a greater extent than most previous ribosome meetings. Third, the meeting congratulated Masayasu Nomura for his important, and still continuing, contributions to the ribosome field.

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 °C with [Mg2+] ≈ 10 mM, P4-P6 folds on the tens of milliseconds timescale with kobs = 15–31 s−1. From these values, an activation free energy ΔG[Dagger] of ∼8–16 kcal/mol is calculated, where the large range for ΔG[Dagger] arises from uncertainty in the pre-exponential factor relating kobs and ΔG[Dagger]. The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes Φ = ΔΔG[Dagger]/ΔΔG°′ are ≈0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The kobs depends on the Mg2+ concentration, and the initial slope of kobs versus [Mg2+] suggests that only ∼1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial ΔG[Dagger] despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.

Non-Watson–Crick base pairs mediate specific interactions responsible for RNA–RNA self-assembly and RNA–protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson–Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2′-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson–Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

The universally conserved A2451 of 23S rRNA has been proposed to participate directly in the catalysis of peptide bond formation in the ribosomal peptidyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid–base catalyst. Increased reactivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH range. Structural data suggested that the interaction between A2451 and G2447 in the ribosome is responsible for A2451 pKa perturbation. In contrast to expectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements in the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2451 interaction is not critical for the ribosome function. We concluded that the proposed unusual pKa of A2451, if existing, may not be crucial for the ribosome activity and that the previously reported pH-dependent alterations in the DMS modification of A2451 do not necessarily reveal an unusual pKa of this nucleotide.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3′ uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21–25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma brucei by expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 × g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20–30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24–26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24–26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24–26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 × 106 copies of hUpf1p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.

The process of eukaryotic gene expression involves a diverse number of steps including transcription, RNA processing, transport, translation, and mRNA turnover. A critical step in understanding this process will be the development of mathematical models that quantitatively describe and predict the behavior of this complex system. We have simulated eukaryotic mRNA turnover in a linear multicomponent model based on the known mRNA decay pathways in yeast. Using rate constants based on experimental data for the yeast unstable MFA2 and stable PGK1 transcripts, the computational modeling reproduces experimental observations after minor adjustments. Subsequent analysis and a series of in silico experiments led to several conclusions. First, we demonstrate that mRNA half-life as commonly measured underestimates the average life span of an mRNA. Second, due to the properties of the pathways, the measurement of a half-life can predominantly measure different steps in the decay network. A corollary of this fact is that different mRNAs will be affected differentially by changes in specific rate constants. Third, the way to obtain the largest change of levels of mRNA for the smallest changes in rate is by changing the rate of deadenylation, where a large amount of regulation of mRNA decay occurs. Fourth, the 3′-to-5′ degradation of mRNA shows mRNA-specific rates of degradation that are dependent on the 5′ structure of the mRNA. These programs can be run over the Web, are adaptable to other eukaryotes, and provide outputs as graphs and virtual northern gels, which can be directly compared to experimental data. Therefore, this model constitutes a useful tool for the quantitative analysis of the process and control of mRNA degradation in eukaryotic cells.

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson–Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min−1. Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

La is an important component of ribonucleoprotein complexes and telomerase is a ribonucleoprotein that compensates for the shortening of the ends of linear DNA by adding telomeric repeats onto the ends of chromosomes by using an integral RNA as the template. We have identified a direct and specific interaction between La and the RNA component of human telomerase. Antibodies specific to La precipitate the human telomerase ribonucleoprotein complex derived from tumor cells, telomerase immortalized normal cells, and in vitro transformed cells. Overexpression of La in both experimentally immortalized human cells and prostate cancer cells results in gradual telomere shortening. Our results demonstrate that La can associate with telomerase and its expression level can influence telomere homeostasis in vivo.

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element G/CGACG/C and also 5′ splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5′ end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5′ splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.

Anticodon hairpins are structural motifs with contradictory functions. The recognition by aminoacyl synthetases implies extended interactions with the anticodon base triplet and thus, usually, an unfolding of the anticodon loop. The recognition by the ribosome and cognate interaction with a mRNA codon implies, on the other hand, the formation of a mini-helix with a canonical anticodon hairpin structure as observed by crystallography and NMR. To be able to understand the various properties of this motif, a precise description of its structural conservation is required. Here, on the basis of phylogenetic, structural, and molecular dynamics data, we discuss a conserved interaction established between the ribose of the U33 and the base at position 35, either a purine or a pyrimidine. This interaction involves the hydrogen bonding donor or acceptor potential of the hydroxyl group of U33 and has to be integrated in an extended definition of the anticodon hairpin. The extended structural signature provides also an explanation for the role played by pseudouridines at position 35.