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.

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.

In the course of analyzing 5′ splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5′ junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5′ junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5′ splice site participates in activation of the cryptic 5′ splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5′ junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5′ splice site even in the presence of a wild-type sequence at the natural 5′ junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5′ splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5′ junction.

In metazoa, a subset of spliceosomal U snRNAs are exported from the nucleus after transcription. This export occurs in a large complex containing a U snRNA, the nuclear cap binding complex (CBC), the leucine-rich nuclear export signal receptor CRM1/Xpo1, RanGTP, and the recently identified phosphoprotein PHAX (phosphorylated adaptor for RNA export). Previous results indicated that PHAX made direct contact with RNA, CBC, and Xpo1 in the U snRNA export complex. We have now performed a systematic characterization of the functional domains of PHAX. The most evolutionarily conserved region of PHAX is shown to be a novel RNA-binding domain that is essential for U snRNA export. In addition, PHAX contains two major nuclear localization signals (NLSs) that are required for its recycling to the nucleus after export. The interaction domain of PHAX with CBC is at least partly distinct from the RNA-binding domain and the NLSs. Thus, the different interaction domains of PHAX allow it to act as a scaffold for the assembly of U snRNA export complexes.

RNase L and Ire1p are members of a superfamily of regulated endoribonucleases that play essential roles in mediating diverse types of cellular stress responses. 2′-5′ oligoadenylates, produced in response to interferon treatment and viral double-stranded RNA, are necessary to activate RNase L. In contrast, unfolded proteins in the endoplasmic reticulum activate Ire1p, a transmembrane serine/threonine kinase and endoribonuclease. To probe their similarities and differences, molecular properties of wild-type and mutant forms of human RNase L and yeast Ire1p were compared. Surprisingly, RNase L and Ire1p showed mutually exclusive RNA substrate specificity and partially overlapping but not identical requirements for phylogenetically conserved amino acid residues in their nuclease domains. A functional model for RNase L was generated based on the comparative analysis with Ire1p that assigns novel roles for ankyrin repeats and kinase-like domains.

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.

Eukaryotic initiation factor (eIF) 4A functions as a subunit of the initiation factor complex eIF4F, which mediates the binding of mRNA to the ribosome. eIF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of eIF4A during translation initiation is to unwind the mRNA secondary structure in the 5′ UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that eIF4A is also required for the translation of mRNAs possessing minimal 5′ UTR secondary structure. Were this hypothesis correct, the requirement for eIF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian eIF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5′ untranslated region are more susceptible to inhibition by the eIF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of eIF4F subunits and eIF4B to the mRNA cap structure is dramatically reduced by the eIF4A mutant and RNA secondary structure. Finally, the eIF4A mutant forms a more stable complex with eIF4G, as compared to the wild-type eIF4A, thus explaining the mechanism by which substoichiometric amounts of mutant eIF4A inhibit translation.

A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random-sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3′,5′-phosphodiester linkage with a catalytic rate of 0.016 min−1. The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3′,5′ linkage with a catalytic rate of 0.32 min−1. The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591–16600). Transesterification proceeds via an in-line mechanism, with the 2′-OH of the bulged nucleotide attacking the 3′-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2′-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2′-oxygen of the bulged nucleotide and the P-O5′ bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2′-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308–1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5′ splice site upstream of the env open reading frame. To determine the role of this splice site in the 5′-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5′ splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5′ splice site, SD4, and the free 5′ end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5′ splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions −3 to +8 of the 5′ splice site and all 11 nt constituting the single-stranded 5′ end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

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.

Nonsense-mediated decay (NMD), also called mRNA surveillance, is an evolutionarily conserved pathway that degrades mRNAs that prematurely terminate translation. To date, the pathway in mammalian cells has been shown to depend on the presence of a cis-acting destabilizing element that usually consists of an exon–exon junction generated by the process of pre-mRNA splicing. Whether or not mRNAs that derive from naturally intronless genes, that is, mRNAs not formed by the process of splicing, are also subject to NMD has yet to be investigated. The possibility of NMD is certainly reasonable considering that mRNAs of Saccharomyces cerevisiae are subject to NMD even though most derive from naturally intronless genes. In fact, mRNAs of S. cerevisiae generally harbor a loosely defined splicing-independent destabilizing element that has been proposed to function in NMD analogously to the spliced exon–exon junction of mammalian mRNAs. Here, we demonstrate that nonsense codons introduced into naturally intronless genes encoding mouse heat shock protein 70 or human histone H4 fail to elicit NMD. Failure is most likely because each mRNA lacks a cis-acting destabilizing element, because insertion of a spliceable intron a sufficient distance downstream of a nonsense codon within either gene is sufficient to elicit NMD.

U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B″ SNF protein, are highly conserved in metazoans.

SR proteins play critical roles in the major pre-mRNA splicing pathway. A second pathway processes U12-dependent AT-AC introns. We demonstrate, by biochemical complementation, the requirement for SR proteins in splicing of AT-AC introns. Whereas SR proteins were sufficient to activate splicing of a P120 AT-AC intron, splicing of a sodium channel AT-AC intron required an additional nuclear fraction. Individual recombinant SR proteins promoted splicing of both substrates, but displayed marked preferences. SR proteins supported basal AT-AC splicing, and also splicing stimulation via a downstream enhancer or conventional 5′ splice site. Analysis of chimeric transcripts revealed that information dispersed throughout exons and introns dictates SR protein specificity and the requirement for the additional nuclear fraction. Thus, SR proteins function in both major and minor splicing pathways, and in coordinating the activities of both spliceosomes via exon definition. These results suggest that despite the substantial differences in intron consensus sequences and in four of the five snRNPs in each spliceosome, at least some of the interactions involving SR proteins are conserved between the two pathways.

The maturation or A-protein gene of single-stranded RNA phage MS2 is preceded by a 130-nt long untranslated leader. When MS2 RNA folding is at equilibrium, the gene is untranslatable because the leader adopts a well-defined cloverleaf structure in which the Shine–Dalgarno (SD) sequence of the maturation gene is taken up in long-distance base pairing with an upstream complementary sequence (UCS). Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand. This requires that formation of the inhibitory cloverleaf is slow. In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed folding is caused by the formation of a metastable intermediate. This intermediate is a small local hairpin that contains the UCS in its loop, thereby preventing or slowing down its pairing with the SD sequence. Mutants in which the small hairpin could not be formed made no detectable amounts of A-protein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small intermediary hairpin was stabilized produced more A-protein than wild type and were viable. One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. The pseudo-revertant had also regained the capacity to produce the A-protein.

Due to printer error, Figure 7A in “A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation” by Tuschl et al. in RNA Vol. 7, No. 1 (pages 29–43) was incorrectly reproduced. Herewith is the entire figure correctly pictured. The Editor and Publisher regret the mistake.