A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473–7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a ρ-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a σA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the ρ-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5′-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3′ end of the gltX transcript and at the 5′ end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA–protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA–protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, “StreptoTag,” should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.

Bob Cedergren passed away on October 14th, his wife Henrietta by his side. For more than seven years, he had fought his cancer tenaciously and optimistically, willingly accepting the rigors of each successive treatment, savoring any respite it offered, however brief, and defying all medical prognostics. He was lucid to the end, his sense of humor intact, directing his laboratory, receiving a stream of visitors, and sharing life as fully as he could with Henrietta, his children Eric and Nellie, and his many friends. In the last years of his life, he transmitted precious lessons to all those around him. He had reached near-perfect serenity and wisdom, and in the most difficult moments it was Bob that supported us with his own inner strength.

The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5′ ends and were complementary to 18S rRNA sequences 609–618 and 1047–1061, homologous to the highly conserved regions designated as the “530 stem-loop” and “790 stem-loop”, respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609–618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047–1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNAPhe.

The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing. The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif. Prior to this study little was known about functional roles for these two domains. We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing. Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing. In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity. Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex. Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p. Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro. The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these. Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix. Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.

An in vitro selection system was devised to select RNAs based on their tertiary structural stability, independent of RNA activity. Selection studies were conducted on the P4–P6 domain from the Tetrahymena thermophila group I intron, an autonomous self-folding unit that contains several important tertiary folding motifs including the tetraloop receptor and the A-rich bulge. Partially randomized P4–P6 molecules were selected based on their ability to fold into compact structures using native gel electrophoresis in the presence of decreasing concentrations of MgCl2. After 10 rounds of the selection process, a number of sequence alterations were identified that stabilized the P4–P6 RNA. One of these, a single base deletion of C209 within the P4 helix, significantly stabilized the P4–P6 molecule and would not have been identified by an activity-based selection because of its essential role for ribozyme function. Additionally, the sequence analysis provided evidence that stabilization of secondary structure may contribute to overall tertiary stability for RNAs. This system for probing RNA structure irrespective of RNA activity allows analysis of RNA structure/function relationships by identifying nucleotides or motifs important for folding and then comparing them with RNA sequences required for function.

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3′ end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3′ end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This ∼125-nt RNA proved to arise via RNase E cleavage from the 3′-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5′ [right arrow] 3′ wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3′ ends act as barriers to 3′ [right arrow] 5′ exonucleolytic mRNA decay.

To understand the RNA-folding problem, we must know the extent to which RNA structure formation is hierarchical (tertiary folding of preformed secondary structure). Recently, nuclear magnetic resonance (NMR) spectroscopy was used to show that Mg2+-dependent tertiary interactions force secondary structure rearrangement in the 56-nt tP5abc RNA, a truncated subdomain of the Tetrahymena group I intron. Here we combine mutagenesis with folding computations, nondenaturing gel electrophoresis, high-resolution NMR spectroscopy, and chemical-modification experiments to probe further the energetic interplay of tertiary and secondary interactions in tP5abc. Point mutations predicted to destabilize the secondary structure of folded tP5abc greatly disrupt its Mg2+-dependent folding, as monitored by nondenaturing gels. Imino proton assignments and sequential NOE walks of the two-dimensional NMR spectrum of one of the tP5abc mutants confirm the predicted secondary structure, which does not change in the presence of Mg2+. In contrast to these data on tP5abc, the same point mutations in the context of the P4–P6 domain (of which P5abc is a subdomain) shift the Mg2+ dependence of P4–P6 folding only moderately, and dimethyl sulfate (DMS) modification experiments demonstrate that Mg2+ does cause secondary structure rearrangement of the P4–P6 mutants' P5abc subdomains. Our data provide experimental support for two simple conclusions: (1) Even single point mutations at bases involved only in secondary structure can be enough to tip the balance between RNA tertiary and secondary interactions. (2) Domain context must be considered in evaluating the relative importance of tertiary and secondary contributions. This tertiary/secondary interplay is likely relevant to the folding of many large RNA and to bimolecular snRNA–snRNA and snRNA–intron RNA interactions.

The rat β-tropomyosin (β-TM) gene encodes both skeletal muscle β-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1–5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3′ splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5′ splice site of exon 7 in nonmuscle cells. The 5′ splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5′ splice site of exon 7 does not result from the sequences at the 3′ end of intron 6 that block the use of the 3′ splice site of exon 7. However, mutating two conserved nucleotides GU at the 5′ splice site of exon 6 results in the efficient use of the 5′ splice site of exon 7. In addition, a mutation that changes the 5′ splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5′ splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first ≈400 residues, whereas the C-terminus provides a nonspecific binding surface.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5′ ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3′ half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5′ exonuclease or endonuclease and a template-directed 3′-to-5′ nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5′ pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.

DNA templates modified with C2′-methoxyls at the last two nucleotides of the 5′ termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single- and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.

Previous studies suggested that domains 5 and 6 (D5 and D6) of group II introns act together in splicing and that the two helical structures probably do not interact by helix stacking. Here, we characterized the major Mg2+ ion- and salt-dependent, long-wave UV light-induced, intramolecular crosslinks formed in 4-thiouridine-containing D56 RNA from intron 5γ (aI5γ) of the COXI gene of yeast mtDNA. Four major crosslinks were mapped and found to result from covalent bonds between nucleotides separating D5 from D6 [called J(56)] and residues of D6 near and including the branch nucleotide. These findings are extended by results of similar experiments using 4-thioU containing D56 RNAs from a mutant allele of aI5γ and from the group IIA intron, aI1. Trans-splicing experiments show that the crosslinked wild-type aI5γ D56 RNAs are active for both splicing reactions, including some first-step branching. An RNA containing the 3-nt J(56) sequence and D6 of aI5γ yields one main crosslink that is identical to the most minor of the crosslinks obtained with D56 RNA, but in this case in a cation-independent fashion. We conclude that the interaction between J(56) and D6 is influenced by charge repulsion between the D5 and D6 helix backbones and that high concentrations of cations allow the helices to approach closely under self-splicing conditions. The interaction between J(56) and D6 appears to be a significant factor establishing a side-by-side (i.e., not stacked) orientation of the helices of the two domains.