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.

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.

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25–99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5′ deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3′ of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.

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.

Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA. The RNAs were passed through a series of rounds of methylation with ErmE. After each round, RNAs were selected that had partially or completely lost their ability to be methylated. After several rounds of methylation/selection, 187 subclones were analyzed. Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051–A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases.

A novel mechanism for the very first step in polypeptide elongation on the ribosome has recently been proposed by Simonson and Lake (2002). According to their transorientation hypothesis, the incoming aminoacyl-transfer RNA (aa-tRNA) initially binds to the messenger RNA (mRNA) with the anticodon stacked on the 5′ side of the anticodon loop; then, following GTP hydrolysis, the tRNA swings into the A-site by switching from the 5′-stacked conformation to the classic 3′-stacked geometry while maintaining Watson–Crick base pairing with the mRNA. There are several serious problems with this proposal.

Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3′ untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA.

Due to a printing error, Figures 3 and 5 of “The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs” by Battle and Doudna in RNA 7-1 (pages 123–132) were reproduced incorrectly, without shaded grey areas. Herewith are the figures correctly pictured. The Editor and Publisher regret the mistake.

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells. Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3′-untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3′ UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3′ UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

The organization of the 5′ terminus region in the 16S rRNA was investigated using a series of RNA constructs in which the 5′ terminus was extended by 5 nt or was shortened to give RNA molecules that started at positions −5, +1, +5, +8, +14, or +21. The structural and functional effects of the 5′ extension/truncations were determined after the RNAs were reconstituted. 30S subunits containing 16S rRNA with 5′ termini at −5, +1, +5, +8 and +14 had similar structures (judged by UV-induced crosslinking) and exhibited a gradual reduction in tRNA binding activity compared to that seen with 30S subunits reconstituted with native 16S rRNA. To create the 5′ terminal site-specific photocrosslinking agent, the reagent azidophenacylbromide (APAB) was attached to the 5′ terminus of 16S rRNA through a guanosine monophosphorothioate and the APA-16S rRNAs were reconstituted. Crosslinking carried out with the APA revealed sites in six regions around positions 300–340, 560, 900, 1080, the 16S rRNA decoding region, and at 1330. Differences in the pattern and efficiency of crosslinking for the different constructs allow distance estimates for the crosslinked sites from nucleotide G9. These measurements provide constraints for the arrangement of the RNA elements in the 30S subunit. Similar experiments carried out in the 70S ribosome resulted in a five- to tenfold lower frequency of crosslinking. This is most likely due to a repositioning of the 5′ terminus upon subunit association.

Positioning rRNA within the ribosome remains a challenging problem. Such positioning is critical to understanding ribosome function, as various rRNA regions interact to form suitable binding sites for ligands, such as tRNA and mRNA. We have used phenanthroline, a chemical nuclease, as a proximity probe, to help elucidate the regions of rRNA that are near neighbors of the stem-loop structure centering at nt 790 in the 16S rRNA of the Escherichia coli 30S ribosomal subunit. Using phenanthroline covalently attached to a DNA oligomer complementary to nt 787–795, we found that nt 582–584, 693–694, 787–790, and 795–797 were cleaved robustly and must lie within about 15 Å of the tethered site at the 5′ end of the DNA oligomer, which is adjacent to nt 795 of 16S rRNA.

In vitro runoff transcription using T7 RNA polymerase has been the method of choice to produce milligram quantities of RNA for structural studies. Unfortunately, the T7 enzyme often adds one or more extra nucleotides at the 3′ end, which results in a heterogeneous RNA product (Milligan et al., 1987). This heterogeneity can be observed at the 5′ end as well, depending on the transcription template (Ferre-D'Amare & Doudna, 1996). The lack of homogeneity, which potentially is deleterious for structural studies, can be overcome with the use of cis- and/or trans-acting ribozymes, which produce clean RNA ends after their catalytic reaction (Ferre-D'Amare & Doudna, 1996). This procedure has been scaled up for large quantities of RNA for NMR and X-ray crystallographic studies, and is useful for purification of larger RNAs (greater than 50 nt) where single-nucleotide resolution by gel electrophoresis is difficult. Ribozymes that have been used in this manner include the hairpin ribozyme, the hammerhead ribozyme (HH), the hepatitis delta ribozyme (δ), and the Neurospora varkud satellite RNA ribozyme (VS) (Guo & Collins, 1995; Price et al., 1995; Ferre-D'Amare & Doudna, 1996). All of these ribozymes leave a 5′-hydroxyl and a 3′-cyclic phosphate as products of cleavage.

The article by Agustín Aranda, José E. Pérez-Ortin, Claire Moore, and Marcel.lí del Olmo, “Transcription termination downstream of the Saccharomyces cerevisiae FPB1 poly(A) site does not depend on efficient 3′ end processing,” that appeared in the March issue of RNA (Vol. 4, No. 3) on pages 303–318 contained a mistake in the title: FPB1 should be FBP1.

“Crystallization, as applied to proteins, was long held in high esteem, but hardly considered science.” I quote from the preface to Alexander McPherson's new survey of the field, Crystallization of Biological Macromolecules. Years ago, McPherson's previous monograph (Preparation and Analysis of Protein Crystals, 1982, Wiley) introduced me to the subject. The growth of knowledge about protein crystallization in the past 17 years is vividly illustrated by comparison of the two books. The slim old volume contained a single chapter on experimental procedures for protein crystallization. In contrast, the 1999 book has three chapters on experimental issues, plus several chapters that review the emerging understanding of the physical chemistry of macromolecular crystallization.

Life today requires the transmission of genetic information in the form of DNA, and a large number of catalytic and structural functions carried out by proteins. Without DNA to specify useful sequences of amino acids, functional proteins would not exist. Without proteins to carry out high-fidelity DNA replication and gene expression, DNA could not serve as the genetic material. How could either arise without the other?

RNA helices that recapitulate sequences of the tRNA acceptor stem, including the 3′ NCCA nucleotides, can be substrates for aminoacyl–tRNA synthetases (Frugier et al., 1994; Hamann & Hou, 1995; Martinis & Schimmel, 1995; Quinn et al., 1995). Although the catalytic efficiency of aminoacylation of RNA helices is reduced from that of the full-length parent tRNA, the specificity is maintained. The specific aminoacylation lies in the ability of aminoacyl–tRNA synthetases to recognize functional groups within the RNA helices. Analysis of tRNA–synthetase structures has suggested a general principle (Rould et al., 1989; Ruff et al., 1991; Arnez & Moras, 1997). The class I synthetases, which attach an amino acid initially to the 2′-OH of the terminal ribose, approach the acceptor and NCCA end from the minor groove side. The class II synthetases, which attach an amino acid to the terminal 3′-OH, approach from the major groove side (Arnez & Moras, 1997). The class-specific approach leads to tRNA–synthetase complexes that are near mirror images of each other and provides a structural rationale for the stereochemistries of aminoacylation. We report here the identification of a functional group in the acceptor end of Escherichia coli tRNACys that is important for the class I cysteine–tRNA synthetase. This functional group makes one of the largest energetic contributions to aminoacylation. However, it is located on the major groove side of the acceptor stem. Kinetic analysis of the contribution of this functional group to aminoacylation suggests new features that are not anticipated from the class-specific approach of synthetases.

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.

We constructed two aptamers, each of which contains a 7-nt-long loop complementary to the anticodon loop of a suppressor tRNA. One of these aptamers can form a stable bimolecular complex with the suppressor tRNA in vitro and protects the 7 nt in the suppressor's anticodon loop from RNase S1. An Escherichia coli strain, carrying an amber mutation in the lac Z gene, produces β-galactosidase only if the suppressor is present; the aptamer's coexpression in the cell inhibits the activity of the suppressor tRNA. Moreover, in E. coli extract, the aptamer partially inhibits the read-through of the stop codon on the part of the suppressor tRNA. These results point to a novel strategy that need not be limited to the suppressor tRNA. By constructing appropriate inducible aptamers, it may well be possible to effectively control translation in vivo.

A cell-free mRNA decay assay has been adapted to permit the kinetics of 3′ → 5′ exoribonuclease activities to be monitored in real time. RNA probes containing 5′ caps and 3′ poly(A) tails generated by transcription in vitro are 3′ labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3′ end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3′ untranslated region and an 85-base poly(A) tail. Following 3′ fluorescein labeling, the rate of 3′-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5′ side of internal loop 1, whereas Prp8p interacts with five different regions of the 5′ stem-loop, but not with the Sm site or 3′ stem-loop. Both internal loops in the 5′ domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5′ stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1–exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.