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.

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.

Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5′-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.

Escherichia coli tRNAVal with pyrimidine substitutions for the universally conserved 3′-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNAVal with a 3′-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3′-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNAVal:EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNAVal for EF-Tu:GTP, thus explaining the inability of these tRNAVal variants to function in polypeptide synthesis. Valyl-tRNAVal terminating in 3′-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3′ end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3′ cytosine. Similar observations were made in studies of the interaction of 3′ end mutants of E. coli tRNAAla and tRNAPhe with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3′ end of aminoacyl-tRNAs.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNAArg and C36 and G37 in tRNAAsp (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069–5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNAArg and tRNAAsp, the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899–4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different kcat and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3′ terminus of the 5′ fragment was ligated to radioactively labeled 4-thiouridine 5′-,3′-biphosphate (“psUp”), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41–42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a “U-turn” in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

A number of heuristic descriptors have been developed previously in conjunction with the mfold package that describe the propensity of individual bases to participate in base pairs and whether or not a predicted helix is “well-determined.” They were developed for the “energy dot plot” output of mfold. Two descriptors, P-num and H-num, are used to measure the level of promiscuity in the association of any given nucleotide or helix with alternative complementary pairs. The third descriptor, S-num, measures the propensity of bases to be single-stranded. In the current work, we describe a series of programs that were developed in order to annotate individual structures with “well-definedness” information. We use color annotation to present the information. The programs can annotate PostScript files that are created by the mfold package or the PostScript secondary structure plots produced by the Weiser and Noller program XRNA (Weiser B, Noller HF, 1995, XRNA: Auto-interactive program for modeling RNA, The Center for Molecular Biology of RNA, Santa Cruz, California: University of California; Internet: ftp://fangio.ucsc.edu/pub/XRNA). In addition, these programs can annotate ss files that serve as input to XRNA. The annotation package can also handle structure comparison with a reference structure. This feature can be used to compare predicted structure with a phylogenetically deduced model, to compare two different predicted foldings, and to identify conformational changes that are predicted between wild-type and mutant RNAs.

We provide several examples of application. Predicted structures of two RNase P RNAs were colored with P-num information and further annotated with comparative information. The comparative model of a 16S rRNA was annotated with P-num information from mfold and with base pair probabilities obtained from the Vienna RNA folding package. Further annotation adds comparisons with the optimal foldings obtained from mfold and the Vienna package, respectively. The results of all of these analyses are discussed in the context of the reliability of structure prediction.

Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, δAg-S and δAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (δAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (δAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3′ end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA–protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.

Substrate sequences surrounding catalytic RNAs but not involved in specific, conserved interactions can severely interfere with in vitro ribozyme activity. Using model group II intron precursor transcripts with truncated or randomized exon sequences, we show that unspecific sequences within the 5′ exon are particularly prone to inhibit both the forward and the reverse first splicing step (branching). Using in vitro selection, we selected efficient 5′ exons for the reverse branching reaction. Precursor RNAs carrying these selected 5′ exons reacted more homogeneously and faster than usual model precursor transcripts. This suggests that unfavorable structures induced by the 5′ exon can introduce a folding step that limits the rate of in vitro self-splicing. These results stress how critical is the choice of the sequences retained or discarded when isolating folding domains from their natural sequence environments. Moreover, they suggest that exon sequences not involved in specific interactions are more evolutionarily constrained with respect to splicing than previously envisioned.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5′ splice site sequence. Here we show that U6atac snRNA also base pairs to the 5′ splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5′ splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson–Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA–RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.