Nucleotide Analog Interference Mapping Research Articles

RNA determinants and protein components of the histone pre‐mRNA processing machinery

Most metazoan mRNAs encoding histones are cleaved but not polyadenylated at their 3′ ends. Cleavage is directed by the U7 small nuclear ribonucleoprotein (U7 snRNP) and requires a heat-labile factor. We recently identified the heat-sensitive complex essential for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor, two subunits of the cleavage stimulation factor and symplekin. To identify which of these factors are essential for cleavage, we are attempting to reconstitute an in vitro processing system with purified and recombinant components. To investigate the role of U7 snRNA in processing, we have determined the RNA requirements for the assembly of a functional U7 snRNP in the Xenopus oocyte system by injecting chimeric histone H4 pre-mRNA / U7 snRNA constructs. We find that the rate limiting step is the assembly of the unique U7 Sm core of proteins, consistent with a role of U7 snRNP in recruiting the additional components of the processing machinery. By Nucleotide Analog Interference Mapping (NAIM) and conventional mutagenesis, we have identified the sequence and structural features of U7 critical for assembly of active particles. These include a flexible 5′-half (revealed by pseudouridine substitution) and four critical nucleotides in the 3′-half of the U7 Sm site, as well as correct placement and length of the U7 hairpin. Research support: HHMI, NIH.

Linking the group II intron catalytic domains: tertiary contacts and structural features of domain 3

Despite its importance for group II intron catalytic activity, structural information on conserved domain 3 (D3) is extremely limited. This domain is known to specifically stimulate the chemical rate of catalysis and to function as a 'catalytic effector'. Of all the long-range tertiary contacts that have been identified within group II introns, none has included D3 residues. Furthermore, little is known about the atoms and functional groups in D3 that contribute to catalysis. Using a nucleotide analog interference mapping assay with an extended repertoire of nucleotide analogs, we have identified functional groups in D3 that are critical for ribozyme activity. These data, together with mutational analysis, suggest the formation of noncanonical base pairs within the phylogenetically conserved internal loop at the base of D3. Finally, a related nucleotide analog interference suppression study resulted in the identification of a direct tertiary interaction between D3 and catalytic domain 5, which sheds new light on D3 function in the group II intron structure and mechanism.

Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein.

The interaction between box C/D small nucleolar (sno)RNAs and the 15.5K protein nucleates snoRNP assembly. Many eukaryotic snoRNAs contain two potential binding sites for this protein, only one of which appears to be utilized in vivo. The binding site conforms to the consensus for a kink-turn motif. We have investigated the molecular basis for selection of one potential site over the other using in vitro mobility shift assays and nucleotide analog interference mapping of Xenopus U25 snoRNA and of a circularly permuted form. We find that preferential binding of human 15.5K is not dependent on the proximity of RNA ends, but instead appears to require a structural context beyond the kink-turn itself. Direct analysis of the energetic contributions to binding made by 18 functional groups within the kink-turn identified both backbone atoms and base functionalities as key for interaction. An intramolecular RNA-RNA contact via a 2'-hydroxyl may supercede a putative Type I A-minor interaction in stabilizing the RNA-protein complex.

Probing RNA Structure and Function by Nucleotide Analog Interference Mapping

Nucleotide analog interference mapping (NAIM) can be used to simultaneously, yet individually, identify structurally or catalytically important functional groups within an RNA molecule. Phosphorothioate-tagged nucleotides and nucleotide analogs are randomly incorporated into an RNA of interest by in vitro transcription. The phosphorothioate tag marks the site of substitution and identifies sites at which the modification affects the structure or function of the RNA molecule. This technique has been expanded to include identification of hydrogen bonding pairs (NAIS), ionizable functional groups, metal ion ligands, and the energetics of protein binding (QNAIM). The analogs, techniques, and data analysis used in NAIM are described here.

Analysis of ribozyme structure and function by nucleotide analog interference mapping.

Nucleotide analog interference mapping (NAIM) is a quick and effective method to define concurrently, yet singly, the importance of specific functional groups at particular nucleotide residues in relation to the structure and function of an RNA. NAIM can be utilized on virtually any RNA with an assayable function, including catalytic RNAs. The method hinges on the ability to successfully incorporate, within an RNA transcript, various 5'-O-(1-thio)nucleoside analogs randomly via in vitro transcription. This can be achieved by using wild-type or Y639F mutant T7 RNA polymerase, thus creating a pool of analog-doped RNAs. When subjected to a selection step to separate the active transcripts from the inactive ones, the pool helps to identify functional groups that are crucial for RNA activity. The technique can be used to study ribozyme structure and function via monitoring of cleavage or ligation reactions, or define functional groups that are critical for RNA folding, RNA-RNA interactions, and RNA interactions with proteins, metals, or other small molecules. All major classes of catalytic RNAs have been examined by NAIM. This is a generalized approach that should provide the scientific community with the tools to better understand the RNA structure-activity relationship (SAR).

Quantitation of free energy profiles in RNA-ligand interactions by nucleotide analog interference mapping.

RNA interactions with protein and small molecule ligands serve a wide variety of biochemical functions in the cell. To best understand the specificity and affinity of these interactions, the free energy contribution made by individual function groups in the RNA must be determined. As an efficient method for obtaining such energetic profiles, we report quantitative nucleotide analog interference mapping (QNAIM). This extension of the NAIM methodology uses the magnitude of analog interference as a function of ligand concentration to calculate binding constants for RNA with individual analog substitutions. In this way, QNAIM not only defines which functional groups are important to an interaction but simultaneously determines the energetic contribution made by each occurrence of that functional group within the RNA polymer. To establish the utility of this approach, QNAIM was used to quantify functional group interactions within the signal recognition particle (SRP), specifically the 4.5S RNA with the M domain of Ffh. In each of the cases in which energetic data were available from previous site-specific substitution analyses, QNAIM provided nearly equivalent results. These experiments on a model system demonstrate that QNAIM is an efficient method to establish a chemically detailed free energy profile for a wide variety of RNA-ligand interactions.

Biochemical detection of adenosine and cytidine ionization within RNA by interference analysis.

Perturbation of active site functional group pKas is an important strategy employed by protein enzymes to achieve catalysis. There is increasing evidence to indicate that RNAs also utilize functional group pKa perturbation for folding and reactivity. The two best candidates for a functionally relevant pKa perturbation are the N3 of C (pKa 4.2) and the N1 of A (pKa 3.5), either of which could be sufficiently raised to allow protonation near physiological pH. Here we report the synthesis and use of a series of alpha-phosphorothioate tagged cytidine and adenosine analogs whose altered pKas make it possible to efficiently detect functionally relevant protonation events by Nucleotide Analog Interference Mapping. This approach has been used to detect ionization events in several catalytic RNAs, including the group I intron, the Hepatitis Delta Virus (HDV), the hairpin and the Varkud Satellite (VS) ribozymes. The active site residue of the VS ribozyme appears to be ionized in the course of the reaction pathway, which may be indicative of a general acid or base mechanism for catalysis by this RNA.

456 Caelyx (pegylated liposomal doxorubicin hcl) and conventional doxorubicin have significantly different adverse event profiles

Despite its small size, the 205 nt group I intron from Azoarcus tRNAIle is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a “chemical phylogeny” of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5′-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2′-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5′-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.

2‘-Mercaptonucleotide Interference Reveals Regions of Close Packing within Folded RNA Molecules

The 2'-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2'-hydroxyl group, we synthesized 2'-mercaptonucleotides for use in nucleotide analogue interference mapping. To correlate the observed interference effects with tertiary structure, we used the independently folding DeltaC209 P4-P6 domain from the Tetrahymena group I intron. We generated populations of DeltaC209 P4-P6 molecules containing 2'-mercaptonucleotides located randomly throughout the domain and separated the folded molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of the RNA molecules revealed the sites at which 2'-mercaptonucleotides interfere with folding. These interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2'-mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed regions within folded RNA molecules.

The functional anatomy of an intrinsic transcription terminator.

To induce dissociation of the transcription elongation complex, a typical intrinsic terminator forms a G.C-rich hairpin structure upstream from a U-rich run of approximately eight nucleotides that define the transcript 3' end. Here, we have adapted the nucleotide analog interference mapping (NAIM) approach to identify the critical RNA atoms and functional groups of an intrinsic terminator during transcription with T7 RNA polymerase. The results show that discrete components within the lower half of the hairpin stem form transient termination-specific contacts with the RNA polymerase. Moreover, disruption of interactions with backbone components of the transcript region hybridized to the DNA template favors termination. Importantly, comparative NAIM of termination events occurring at consecutive positions revealed overlapping but distinct sets of functionally important residues. Altogether, the data identify a collection of RNA terminator components, interactions and spacing constraints that govern efficient transcript release. The results also suggest specific architectural rearrangements of the transcription complex that may participate in allosteric control of intrinsic transcription termination.

Domains 2 and 3 Interact to Form Critical Elements of the Group II Intron Active Site

Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5γ group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5′-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j2/3 linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5′-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known θ–θ′ interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site.

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.

Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease.

RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.

Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA.

Nucleotide analog interference mapping (NAIM) is a powerful method for identifying RNA functional groups involved in protein-RNA interactions. We examined particles assembled on modified U1 small nuclear RNAs (snRNAs) in vitro and detected two categories of interferences. The first class affects the stability of two higher-order complexes and comprises changes in two adenosines, A65 and A70, in the loop region previously identified as the binding site for the U1 small nuclear ribonucleoprotein (snRNP)-specific U1A protein. Addition of an exocyclic amine to position 2 of A65 interferes strongly with protein binding, whereas removal or modification of the exocyclic amine at position 6 makes little difference. Modifications of A70 exhibit the opposite effects: Additions at position 2 are permitted, but modification of the exocyclic amine at position 6 significantly inhibits protein binding. These interactions, critical for U1A-U1 snRNA recognition in the context of in vitro snRNP assembly, are consistent with previous structural studies of the isolated protein with the RNA hairpin containing the U1A binding site. The second category of interferences affects all partially assembled U1-protein complexes by decreasing the stability of Sm core protein associations. Interestingly, most strong interferences occur at phosphates in the terminal stem-loop region of U1, rather than in the Sm binding site. These data argue that interactions with the phosphate backbone of the terminal stem loop are essential for the stable association of Sm core proteins with the U1 snRNA. We suggest that the stem loop of all Sm snRNAs may act as a clamp to hold the ring of Sm proteins in place.

Exclusive Interaction of the 15.5 kD Protein with the Terminal Box C/D Motif of a Methylation Guide snoRNP

Box C/D small nucleolar RNAs (snoRNAs) direct site-specific methylation of ribose 2′-hydroxyls in ribosomal and spliceosomal RNAs. To identify snoRNA functional groups contributing to assembly of an active box C/D snoRNP in Xenopus oocytes, we developed an in vivo nucleotide analog interference mapping procedure. Deleterious substitutions consistent with requirements for binding the 15.5 kD protein clustered within the terminal box C/D motif only. In vitro analyses confirmed a single interaction site for recombinant 15.5 kD protein and identified the exocyclic amine of A89 in box D as essential for binding. Our results argue that the 15.5 kD protein interacts asymmetrically with the two sets of conserved box C/D elements and that its binding is primarily responsible for the stability of box C/D snoRNAs in vivo.

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.

The contribution of 2'-hydroxyls to the cleavage activity of the Neurospora VS ribozyme.

We have used nucleotide analog interference mapping and site-specific substitution to determine the effect of 2'-deoxynucleotide substitution of each nucleotide in the VS ribozyme on the self-cleavage reaction. A large number of 2'-hydroxyls (2'-OHs) that contribute to cleavage activity of the VS ribozyme were found distributed throughout the core of the ribozyme. The locations of these 2'-OHs in the context of a recently developed helical orientation model of the VS ribozyme suggest roles in multi-stem junction structure, helix packing, internal loop structure and catalysis. The functional importance of three separate 2'-OHs supports the proposal that three uridine turns contribute to local and long-range tertiary structure formation. A cluster of important 2'-OHs near the loop that is the candidate region for the active site and one very important 2'-OH in the loop that contains the cleavage site confirm the functional importance of these two loops. A cluster of important 2'-OHs lining the minor groove of stem-loop I and helix II suggests that these regions of the backbone may play an important role in positioning helices in the active structure of the ribozyme.

PK(a) perturbation in genomic Hepatitis Delta Virus ribozyme catalysis evidenced by nucleotide analogue interference mapping.

The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pK(a) perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs, in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.

Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme

We have detected by nucleotide analog interference mapping (NAIM) AMPαS and IMPαS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit. Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II). Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b). Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes. Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 Å from each other in the current 3D model of a full-length RNase P RNA-substrate complex. Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 Å, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture. Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a. Alternatively, based on the recent finding that B. subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II.

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).