M1 RNA that contained 4′-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA–substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (Δ[273–281]), but not for the M1 RNA–product complex. High concentrations of Mg2+ increased the yield of all M1 RNA–substrate complexes but not the M1 RNA–product complex.

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3′-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.

In the natural form of the hairpin ribozyme the two loop-carrying duplexes that comprise the majority of essential bases for activity form two adjacent helical arms of a four-way RNA junction. In the present work we have manipulated the sequence around the junction in a way known to perturb the global folding properties. We find that replacement of the junction by a different sequence that has the same conformational properties as the natural sequence gives closely similar reaction rate and Arrhenius activation energy for the substrate cleavage reaction. By comparison, rotation of the natural sequence in order to alter the three-dimensional folding of the ribozyme leads to a tenfold reduction in the kinetics of cleavage. Replacement with the U1 four-way junction that is resistant to rotation into the antiparallel structure required to allow interaction between the loops also gives a tenfold reduction in cleavage rate. The results indicate that the conformation of the junction has a major influence on the catalytic activity of the ribozyme. The results are all consistent with a role for the junction in the provision of a framework by which the loops are presented for interaction in order to create the active form of the ribozyme.

Ro RNPs are small cytoplasmic RNA–protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264–273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5′/3′ terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122–10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5′/3′ double-stranded stem region that is conserved between different Y RNAs.

The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain [Alu domain homolog in yeast (Adhy)], we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA. Srp14p binds as a homodimeric complex to the 5′ sequences of scR1 RNA. Its minimal binding site consists of 99 nt (Adhy RNA), comprising a short hairpin structure followed by an extended stem. As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies. In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins. These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function. Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA–protein recognition as well as in the overall structure of the complex. The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms.

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24–rRNA–L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA–protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5′ and 3′ splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5′ splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

The conjugative transfer of F-plasmids is repressed by a two-component system, which consists of the antisense RNA FinP and the protein FinO. FinO binds FinP, protecting it from endonucleolytic degradation and facilitating duplex formation between FinP and its complementary RNA. Here we present the results of site-specific protein–RNA crosslinking and gel-based fluorescence resonance energy transfer (gelFRET) experiments used to probe the structure of a complex of FinO bound to an RNA target consisting of a duplex with 5′ and 3′ single-stranded tails. The crosslinking experiments reveal that an extensive, largely positively charged surface on FinO contacts RNA. The gelFRET measurements indicate that the 5′ single-stranded tail of the RNA is in closer contact with much of the protein than the distal, blunt end of the RNA duplex. These data suggest that significant conformational adjustments in the protein and/or the RNA accompany complex formation.

Group I introns constitute excellent systems for analyzing the relationship between RNA tertiary folding and catalysis. Within a hierarchical framework interpretation of RNA folding, secondary structure motifs subtend RNA three-dimensional (3D) architecture. Thus, mutations in two-dimensional motifs are expected to have effects different from those disrupting 3D contacts. Using UV spectroscopy, we have studied the influence of nucleotide substitutions, in both secondary and tertiary structure elements, on the thermal stability of the tertiary folding of the bacteriophage T4 td group I intron. Further, we present a quantitative analysis of the relationship between the splicing efficiency in vivo and the stability of the intron structure as monitored by UV melting curves. We conclude that the stability of the tertiary structure of a group I intron as measured by UV melting is generally a good indication of its ability to splice in vivo.

Human RNase P recognizes a small model substrate consisting of only the 5′ leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58–60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55–57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2′-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.

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.

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.

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.

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.

P5abc domain of Tetrahymena LSU intron functions as an activator that is not essential for but enhances the activity of the ribozyme either when present in cis or when added in trans. This domain contains three regions (A-rich bulge, L5b, and L5c) that have been demonstrated to interact with the rest of the intron. Although these regions are presumably important for efficient activation, the role of each element is not understood in the mechanism of activation. We employed circularly permuted introns and examined the roles of each element. The results show that each of the three elements can activate the intron independently. We also found that a correlation between the activation by P5abc and the physical affinity of P5abc to the intron exists.

SRrp86 is an 86-kDa member of the SR protein superfamily that is unique in that it can alter splice site selection by regulating the activity of other SR proteins. To study the function of SRrp86, inducible cell lines were created in which the concentration of SRrp86 could be varied and its effects on alternative splicing determined. Here, we show that SRrp86 can activate SRp20 and repress SC35 in a dose-dependent manner both in vitro and in vivo. These effects are apparently mediated through direct protein–protein interaction, as pull-down assays showed that SRrp86 interacts with both SRp20 and SC35. Consistent with the hypothesis that relatively modest changes in the concentration or activity of one or more splicing factors can combinatorially regulate overall splicing, protein expression patterns of SRrp86, SRp20, and SC35 reveal that each tissue maintains a unique ratio of these factors. Regulation of SR protein activity, coupled with regulated protein expression, suggest that SRrp86 may play a crucial role in determining tissue specific patterns of alternative splicing.

Unlike most eukaryotic mRNAs studied to date, Xenopus serum albumin mRNA has a short (17-residue), discrete poly(A) tail. We recently reported that this short poly(A) tail results from regulation of the length of poly(A) on albumin pre-mRNA. The purpose of the present study was to locate the cis-acting element responsible for this, the poly(A)-limiting element or PLE. An albumin minigene consisting of albumin cDNA joined in exon 13 to the 3′ end of the albumin gene produced mRNA with <20 nt poly(A) when transfected into mouse fibroblasts. This result indicates both that cis-acting sequences that regulate poly(A) length are within this construct, and that nuclear regulation of poly(A) length is conserved between vertebrates. Poly(A) length regulation was retained after replacing the terminal 53 bp and 3′ flanking region of the albumin gene with a synthetic polyadenylation element (SPA). Conversely, fusing albumin gene sequence spanning the terminal 53 bp of the albumin gene and 3′ flanking sequence onto the human β-globin gene yielded globin mRNA with a 200-residue poly(A)tail. These data indicate that the PLE resides upstream of the sequence elements involved in albumin pre-mRNA 3′ processing. Poly(A) length regulation was restored upon fusing a segment bearing albumin intron 14, exon 15, and 3′ flanking sequence onto the β-globin gene. We demonstrate that exon 15 contains two PLEs that can act independently to regulate the length of poly(A).

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein–protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5′ and 3′ splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3′ splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3′AG recognition. Indeed, deletion of SKY1 specifically suppressed 3′AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3′AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.

Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-U1A and U2 snRNP-U2B″. The structure and sequence of SNF, U1A, and U2B″ are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B″ binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf 's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the “RNA-specificity” domain is necessary for SNF's dual function whereas the mixed motif in the U2A′-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.