RNase MRP and RNase P are both ribonucleoprotein enzymes performing endonucleolytic cleavage of RNA. RNase MRP cleaves at a specific site in the precursor-rRNA transcript to initiate processing of the 5.8S rRNA. RNase P cleaves precursor tRNAs to create the 5′ end of the mature tRNAs. In spite of their different specificities, the two RNases have significant structural similarities. For example, the two enzymes in Saccharomyces cerevisiae share eight protein subunits; only one protein is unique to each enzyme. The RNA components of the two nucleases also show striking secondary-structure similarity. To begin to characterize the role of the RNA subunits in enzyme function and substrate specificity, we swapped two hairpin structures (MRP3 and P3) between RNase MRP RNA and RNase P RNA of S. cerevisiae. The hairpins in the two enzymes could be exchanged without loss of function or specificity. On the other hand, when the MRP3 hairpin in RNase MRP of S. cerevisiae was replaced with the corresponding hairpin from the RNA of Schizosaccharomyces pombe or human RNase MRP, no functional enzyme was assembled. We propose that the MRP3 and P3 hairpins in S. cerevisiae perform similar functions and have coevolved to maintain common features that are different from those of MRP3 and P3 hairpins in other species.

Subcellular localization signals for several mRNAs are positioned in their 3′ untranslated regions (UTR). We have utilized the human α- and β-actin 3′ UTRs as signals for colocalizing hammerhead ribozymes with a lacZ target mRNA. Ribozyme and target genes containing matched or unmatched 3′ UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. β-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl β-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched α- or β-actin 3′ UTR (α–α or β–β) was enhanced approximately threefold relative to unmatched 3′ UTRs. The increase in ribozyme-mediated inhibition of β-galactosidase activity observed when matched 3′ UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.

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.

The process of mRNA splicing is sensitive to in vivo thermal inactivation, but can be protected by pretreatment of cells under conditions that induce heat-shock proteins (Hsps). This latter phenomenon is known as “splicing thermotolerance”. In this article we demonstrate that the small nuclear ribonucleoprotein particles (snRNPs) are in vivo targets of thermal damage within the splicing apparatus in heat-shocked yeast cells. Following a heat shock, levels of the tri-snRNP (U4/U6.U5), free U6 snRNP, and a pre-U6 snRNP complex are dramatically reduced. In addition, we observe multiple alterations in U1, U2, U5, and U4/U6 snRNP profiles and the accumulation of precursor forms of U4- and U6-containing snRNPs. Reassembly of snRNPs following a heat shock is correlated with the recovery of mRNA splicing and requires both Hsp104 and the Ssa Hsp70 family of proteins. Furthermore, we correlate splicing thermotolerance with the protection of a subset of snRNPs by Ssa proteins but not Hsp104, and show that Hsp70 directly associates with U4- and U6-containing snRNPs in splicing thermotolerant cells. In addition, our results show that Hsp70 plays a role in snRNP assembly under normal physiological conditions.

An in vitro selection was designed to identify RNA-cleaving ribozymes predisposed for function as a drug. The selection scheme required the catalyst to be trans-acting with phosphodiesterase activity targeting a fragment of the Kras mRNA under simulated physiological conditions. To increase stabilization against nucleases and to offer the potential for improved functionality, modified sequence space was sampled by transcribing with the following NTPs: 2′-F-ATP, 2′-F-UTP, or 2′-F-5-[(N-imidazole-4-acetyl) propylamine]-UTP, 2′-NH2-CTP, and GTP. Active motifs were identified and assessed for their modified NMP and divalent metal dependence. The minimization of the ribozyme's size and the ability to substitute 2′-OMe for 2′-F and 2′-NH2 moieties yielded the motif from these selections most suited for both nuclease stability and therapeutic development. This motif requires only two 2′-NH2-Cs and functions as a 36-mer. Its substrate sequence requirements were determined to be 5′–Y-G-H–3′. Its half-life in human serum is >100 h. In physiologically relevant magnesium concentrations [∼1 mM] its kcat = 0.07 min−1, Km = 70 nM. This report presents a novel nuclease stable ribozyme, designated ZinzymeTM, possessing optimal activity in simulated physiological conditions and ready for testing in a therapeutic setting.

Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916–923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926–1936). However, we observed that a U1 5′ splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915–926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U11 5′ splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.

Eukaryotic nucleoli contain a large family of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs) that are involved in processing and site-specific methylation of pre-rRNA. Several proteins have been reported to be common factors of box C/D snoRNPs in lower and higher eukaryotes; nevertheless none of them has been clearly shown to directly interact with RNA. We previously identified in Xenopus laevis, by means of UV crosslinking in vivo, two proteins associated with box C/D snoRNAs, fibrillarin and p68. Here we show that fibrillarin interacts directly and specifically with the U16 box C/D snoRNA in a X. laevis oocyte nuclear extract and that it does not require p68 for binding. Specific binding is also obtained with a recombinant fibrillarin demonstrating that the protein is able to bind directly and specifically to U16 snoRNA by itself.

To identify splicing factors in proximity of the 5′ splice site (5′SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5′SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5′SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5′SS:U1 snRNA duplex, and at the same time as a 5′SS translocase, which, upon NTP-dependent conformational change, positions the 5′SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5′SS takes place regardless of whether the 5′SS is originally duplexed with U1 snRNA.

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 5′ domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.

Only 40 of the 359 nucleotides of Potato Spindle Tuber Viroid (PSTVd) represent the virulence-modulating (VM) region. Minor sequence variations in this domain distinguish mild from severe and even necrotic strains. Our recent hypothesis (Owens RA et al., 1996, Virology 222:144–158) that these differences result in varying degrees of bending of this part of the molecule could be tested experimentally. By in vitro transcription and partial double-strand formation, three types of model RNAs were prepared and subjected to electrophoresis in polyacrylamide gels: (1) Fragments representing the VM regions of six different PSTVd strains; (2) control fragments containing a bulge-loop as a rigid bend or an internal loop as a point of increased flexibility; and (3) dsRNAs of 36, 39, and 43 bp as length standards. Migration anomalies in gels of increasing percentage were evaluated and resulted in the following conclusions. In the absence of Mg2+, the VM regions differ only in terms of flexibility. Addition of Mg2+ induces conformational changes in these RNAs. All strains but Mild exhibit a rigid bend, and the angle of bending increases monotonically with the pathogenicity of the strain. The data are discussed in terms of a mechanism of pathogenicity, that protein-binding to the VM region is the primary pathogenic event.

Transcriptional regulation of the T box family of aminoacyl–tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specific interaction with the cognate uncharged tRNA. The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a “specifier sequence” in the leader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator. Previous studies have indicated that although these interactions are necessary for antitermination, they are unlikely to be sufficient. In the current study, the effect of multiple mutations in tRNATyr on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations in helical regions of tRNATyr that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not. The long variable arm of tRNATyr could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure. These results indicate that the tRNA–leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.

The observation that cells restrict the nuclear export of incompletely spliced transcripts via the canonical nuclear mRNA export pathway implies that all retroviruses should have evolved a way to direct the unspliced form of their genomic RNA into an alternate export pathway. While the Crm1-dependent pathway used by complex retroviruses to export incompletely spliced viral transcripts is now fairly well understood, less is known about how simple retroviruses accomplish this task. However, the Mason–Pfizer monkey virus (MPMV) has been shown to encode a structured RNA sequence, termed the constitutive transport element (CTE), that recruits a cellular RNA export factor termed Tap. Here we demonstrate that a CTE previously proposed to be present in the avian sarcoma/leukemia (ASV/ALV) family of retroviruses indeed functions as a potent RNA export signal. We have mapped single- and double-stranded regions present in the ASV/ALV CTE in vitro and report that this CTE is predicted to fold into a structure bearing three distinct RNA stem-loops. However, only the central stem-loop is critical for CTE function and this 69-nt structure is, in fact, sufficient when present as a dimer. While the ASV/ALV CTE is shown to function independently of Crm1, as also previously reported for the MPMV CTE, it lacks any evident sequence homology to the highly conserved MPMV CTE sequence. Together, these data define the secondary structure and biological activity of an avian CTE sequence that may access a novel nuclear RNA export pathway.

The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA–protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3′,5′-phosphodiester linkage at the cleavage site; however, a 2′,5′-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2′,3′-cyclic phosphate and 5′-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3′,5′-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2′,5′-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2′,5′-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5′ to the cleavage site.

Splicing factor SF1 contributes to the recognition of the 3′ splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E. These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce). The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins. These domains mediate protein–protein or protein–RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes. Injection of double-stranded RNA into C. elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan.

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.

The tRNA 3′ end contains the conserved CCA sequence at the 74–76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74–76 positions was investigated using synthetic minihelix substrates that contain permuted 3′ ends. Results here indicate that the enzyme has the ability to synthesize unusual 3′ ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.

The trpRNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2–3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP–RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5′-(UAGCC)-3′ repeated 11 times and measured their effect on the TRAP–RNA interaction. Deoxyribonucleoside substitutions revealed that a 2′-hydroxyl group (2′-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP–RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2′-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.

A new RNA structural motif consisting of two double helices closely packed via minor grooves is found in many places in the ribosome structure. The packing requires that a GU base pair in one helix be packed against a Watson–Crick pair in the other helix. Two such motifs mediate the interaction of the P- and E-tRNA with the large ribosomal subunit. Analysis of the particular positions of these two motifs in view of the available data on occupancy of tRNA-binding sites and structural changes in the ribosome during the elongation cycle suggests a distinct role for each motif in tRNA translocation.