Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

Selenocysteine biosynthesis and its cotranslational incorporation into selenoproteins are achieved by a complex molecular machinery (reviewed in Hüttenhofer & Böck, 1998). A major wheel in this mechanism is the tRNASec, which plays a pivotal role. It is first charged with serine by the conventional SerRS, the seryl-residue being further converted in situ to the selenocysteyl-residue by the selenocysteine synthase enzyme. The charged selenocysteyl-tRNASec delivers selenocysteine to the nascent polypeptide chain in response to a reprogrammed UGA codon. The classical elongation factors EF-Tu (in bacteria) or EF1-α (in eukaryotes) do not intervene at this stage. Instead, this process requires a selenocysteine-specific translation factor, called SELB in bacteria, but for which no eukaryotic homologue has been cloned yet. Interestingly, antideterminants against EF-Tu binding were found in the Escherichia coli tRNASec (Rudinger et al., 1996). Based on the several functions that this tRNA has to accomplish, it is reasonable to expect that the tRNASec secondary structure should exhibit distinctive structural features deviating from classical elongator tRNAs. In this regard, functional studies, structural probing, and sequence comparisons confirmed the earlier proposal that the bacterial tRNASec needs an 8-bp long amino acceptor stem and a 6-bp long D-stem to function, instead of the canonical 7 bp and 3/4 bp, respectively (Leinfelder et al., 1988; Baron et al., 1990, 1993; Tormay et al., 1994).

The CCA end is common to all tRNAs as the universal site for amino acid attachment. It is also conserved in the 3′-terminal tRNA-like structure of viral genomes that can be aminoacylated by an aminoacyl-tRNA synthetase (Florentz & Giegé, 1995). During aminoacylation, the CCA end enters the catalytic center of an aminoacyl-tRNA synthetase and provides the site for chemistry to take place. The CCA end is also widely used in replication of retroviruses, the bacterial single-stranded RNA viruses, and duplex DNA plasmids of fungal mitochondria. During replication, the CCA end interacts with the template-specificity domain of reverse transcriptase or replicase and provides the initiation site for primer binding and extension (Maizels & Weiner, 1994). The importance of the CCA end in translation and in replication suggests that its conformation will play a role in these two fundamental processes.

Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.

Our knowledge of RNA structure and function has progressed at an astonishingly fast pace in the last five years. Since then, we have seen the X-ray structure of a full ribozyme, the awesome structure of a large domain of a group I intron, and recently the striking structure of the loop E of 5S rRNA. NMR studies have yielded structures, not only of small recurrent loops, but also of several RNA aptamers and of a fragment of the 16S rRNA complexed with an antibiotic. Both methods have also contributed enormously to our understanding of RNA–protein interactions; the richness of the tRNA synthetase interactions is now recognized, whereas the structures of complexes from the spliceosome and the ribosome point to new horizons. The 22 chapters of this new book compose a tribute to the fascinating and ubiquitous versatility of the RNA molecule.

The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5′ UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5′ UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under noncompetitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5′ UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.

The hammerhead ribozyme is a small RNA motif that self cleaves at a specific phosphodiester bond to produce 2′,3′ cyclic phosphate and 5′ hydroxyl termini (Hutchins et al., 1986; Forster & Symons, 1987a). The secondary structure of the hammerhead consists of three helices of arbitrary sequence and length (designated I, II, and III) that intersect at 15 nucleotides termed the catalytic core (Fig. 1A) (Forster & Symons, 1987b; Hertel et al., 1992). The X-ray crystal structures of two hammerhead ribozyme–inhibitor complexes revealed that the core residues fold into two separate domains and the helices are arranged in a Y-shape conformation with helix I and helix II forming the upper portion of the Y (Pley et al., 1994; Scott et al., 1995). Although the hammerhead is found as an intramolecular motif embedded in several RNAs in vivo (Symons, 1989), it can be assembled from two separate oligonucleotides (Fig. 1B) in three different arrangements (Uhlenbeck, 1987; Haseloff & Gerlach, 1988; Koizumi et al., 1988; Jeffries & Symons, 1989). In these bimolecular formats, the hammerhead effects RNA cleavage in a similar manner to a true “enzyme,” proceeding through multiple rounds of substrate binding, cleavage, and product release (Uhlenbeck, 1987).

Insertion of selenocysteine into a growing peptide requires the unusual tRNASec (Zinoni et al., 1987; Stadtman, 1990; Böck et al., 1991). This tRNA has an extended D-stem containing six base pairs, which, in the case of eukaryotic tRNASec (euk-tRNASec), is the key identity element for selenylation and phosphorylation (Wu & Gross, 1994; Amberg et al., 1996). Two secondary structures have been proposed for the euk-tRNASec, which differ in the base pairing of the acceptor/T helical domain (Diamond et al., 1981; Böck et al., 1991; Sturchler et al., 1993). One structure has the normal seven base pairs in the acceptor stem and five base pairs in the T-stem (7/5 structure, Fig. 1, left), and is characterized by an unusually long four-nucleotide unpaired region between the acceptor and D-stems (Connector 1) and an unpaired nucleotide, C64a, in the T-stem. The alternate structure features the normal two nucleotides in Connector 1 and a 13-base pair acceptor/T domain comprised of nine base pairs in the acceptor stem and four in the T-stem (9/4 structure, Fig. 1, right). This 9/4 structure was initially proposed by analogy with the prokaryotic tRNASec (prok-tRNASec), which also contains 13 base pairs in the acceptor/T helical domain. However, in this case, there are eight and five base pairs in the acceptor and T-stems, respectively. The acceptor/T helical domain having 13 base pairs is thought to be a key structural element determining the functionalities pattern of tRNASec in both prokaryotes and eukaryotes (Böck et al., 1991).

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 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160–200, 320, and 340–350 in the 5′ domain, and to positions 1427–1430 and 1439–1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.

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.

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.

We are using the rat β-tropomyosin (β-TM) gene as a model system to study the mechanism of alternative splicing. Previous studies demonstrated that the use of the muscle-specific exon is associated with the use of distant branch points located 147–153 nt upstream of the 3′ splice site. In addition, at least one protein, the polypyrimidine tract binding protein (PTB), specifically interacts with critical cis-acting sequences upstream of exon 7 that are involved in blocking the use of this alternative exon in nonmuscle cells. In order to further study the role of PTB, monoclonal antibodies to PTB were prepared. Anti-PTB antibodies did not inhibit the binding of PTB to RNA because they were able to supershift RNA–PTB complexes. To determine if additional proteins interact with sequences within the pre-mRNA, 35S-met-labeled nuclear extracts from HeLa cells were mixed with RNAs and the RNA–protein complexes were recovered by immunoprecipitation using antibodies to PTB. When RNAs containing intron 6 were added to an 35S-met-labeled nuclear extract, precipitation with PTB antibodies showed a novel set of proteins. By contrast, addition of RNAs containing introns 5 or 7 gave the same results as no RNA, indicating that these RNAs are unable to form stable complexes with PTB. These results are in agreement with our previous studies demonstrating that PTB interacts with sequences within intron 6, but not with sequences within introns 5 and 7. When 35S-met-labeled HeLa nuclear extracts were mixed with biotinylated RNA containing intron 6 and the RNA–protein complexes were recovered using streptavidin-agarose beads, an identical pattern of proteins was observed when compared with the immunoprecipitation assay. Analysis of the proteins that assembled on introns 5, 6, or 7 using biotinylated RNA revealed a unique set of proteins that interact with each of these sequences. The composition of proteins interacting with sequences associated with the use of the 3′ splice site of intron 6 included proteins of 30, 40, 55, 60, 65, 70, 80, and 100 kDa. Microsequencing identified two of the proteins to be Sam68 and the Far Upstream Element Binding Protein (FBP) from the c-myc gene. In addition, a comparison of the proteins that assemble on introns from the α- and β-TM genes that utilize distant branch points revealed common as well as unique proteins that assemble on these introns. These studies identify a set of proteins, in addition to PTB, that are likely involved in the use of distant branch sites associated with the use of alternatively spliced introns.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5′ untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5′ UTR of msl-2 mRNA resulted in retention of the 5′ UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5′ and 3′ splice sites as well as the deoxyribose substitution of the ribose 2′ hydroxyl group at the 5′ splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5′ splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3′ splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5′ and 3′ splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.

NMR has been used to examine the conformational properties of two variants of the sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, which is essential for elongation factor interactions with the ribosome: (1) its bacterial homologue, which lacks two of the bases that flank the conserved 12-nt sequence in the middle of the SRL, but which is functionally equivalent, and (2) a functionally active variant of the eukaryotic SRL in which the bulged G within the conserved sequence is replaced by an A. The data indicate that, although the bacterial SRL is less stable than the eukaryotic SRL, its conformation is closely similar. Furthermore, even though replacement of the bulged G in the SRL with an A seriously destabilizes the center of the loop, its effect on the overall conformation of the SRL appears to be modest. In the course of this work, it was serendipitously discovered that at neutral pH, the C8 proton of the bulged G, in both PRO-SRL and E73, exchanges about 10 times faster than it does in GMP.

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.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor–substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor–cargo interaction and imparts directionality to the transport process.

Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc Nat Acad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure–function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure–function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.