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PubMed2003

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

Base Sequence↗
PubMed1999

5' splice site mutations in tau associated with the inherited dementia FTDP-17 affect a stem-loop structure that regulates alternative splicing of exon 10.

Missense and splice site mutations in the microtubule-associated protein tau gene were recently found associated with fronto-temporal dementia and parkinsonism linked to chromosome 17 (Poorkaj et al. (1998) Ann. Neurol. 43, 815-825; Hutton et al. (1998) Nature 393, 702-705; Spillantini et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7737-7741). The mutations in the 5' splice site of exon 10 were shown to increase the ratio of tau mRNAs containing exon 10 and thus the proportion of Tau protein isoforms with 4 microtubule binding repeat domains, although how this increase leads to neurodegeneration is presently unclear. The mechanism by which these mutations increase tau exon 10 splicing was not determined, although the mutations were predicted to disrupt a potential stem-loop structure that was likely involved in the regulation of exon 10 alternative splicing. Here we describe in vitro splicing assays and RNA structural analysis that demonstrate that the mutations do indeed act through disruption of the stem-loop structure and that the stability of this secondary structure feature at least partially determines the ratio of tau exon 10+/- transcripts. In addition, we provide evidence that the stability of the stem-loop structure underlies the alternative splicing of this exon in other species.

Alternative Splicing↗
PubMed1998

Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets.

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.

Amino Acid Sequence↗
PubMed2001

The thermodynamic origin of the stability of a thermophilic ribozyme.

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg(2+) to fold to their native structures that are very similar. The stability is measured as a function of Mg(2+) and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Base Sequence↗
PubMed1994

Modeling large RNAs and ribonucleoprotein particles using molecular mechanics techniques.

There is a growing body of low-resolution structural data that can be utilized to devise structural models for large RNAs and ribonucleoproteins. These models are routinely built manually. We introduce an automated refinement protocol to utilize such data for building low-resolution three-dimensional models using the tools of molecular mechanics. In addition to specifying the positions of each nucleotide, the protocol provides quantitative estimates of the uncertainties in those positions, i.e., the resolution of the model. In typical applications, the resolution of the models is about 10-20 A. Our method uses reduced representations and allows us to refine three-dimensional structures of systems as big as the 16S and 23S ribosomal RNAs, which are about one to two orders of magnitude larger than nucleic acids that can be examined by traditional all-atom modeling methods. Nonatomic resolution structural data--secondary structure, chemical cross-links, chemical and enzymatic footprinting patterns, protein positions, solvent accessibility, and so on--are combined with known motifs in RNA structure to predict low-resolution models of large RNAs. These structural constraints are imposed on the RNA chain using molecular mechanics-type potential functions with parameters based on the quality of experimental data. Surface potential functions are used to incorporate shape and positional data from electron microscopy image reconstruction experiments into our models. The structures are optimized using techniques of energy refinement to get RNA folding patterns. In addition to providing a consensus model, the method finds the range of models consistent with the data, which allows quantitative evaluation of the resolution of the model. The method also identifies conflicts in the experimental data. Although our protocol is aimed at much larger RNAs, we illustrate these techniques using the tRNA structure as an example and test-bed.

Anticodon↗
PubMed1984

Ribosomal proteins: their structure and spatial arrangement in prokaryotic ribosomes.

During the last 15 years of ribosomal protein study, enormous progress has been made. Each of the proteins from E. coli ribosomes has been isolated, sequenced, and immunologically and physically characterized. Ribosomal proteins from other sources (e.g., from some bacteria, yeast, and rat) have been isolated and studied as well. Several proteins have recently been crystallized, and from the X-ray studies it is expected that much important information on the three-dimensional structure will be forthcoming. Many other proteins can probably be crystallized if suitable preparative procedures and crystallization conditions are found. Tremendous progress has also been made in deciphering the architecture of the ribosome. A battery of different methods has been used to provide the nearest neighbor distances of the ribosomal proteins in situ. Definitive measurements are now emanating from neutron-scattering experiments which also promise to give reasonably accurate radii of gyration of the proteins in situ. In turn, refined immune electron microscopy results supplement the neutron-scattering data and also position the proteins on the subunits themselves. This cannot be done by the other methods. Determination of the three-dimensional RNA structure within the ribosome is still in its infancy. Nonetheless, it is expected that by combining the data from protein-RNA and from RNA-RNA cross-linking studies, the structure of the RNA in situ can be unraveled. Of great interest is the fact that ribosomal subunits and ribosomes themselves have now been crystallized, and low-resolution structural maps have already been obtained. However, to grow suitable crystals and to resolve the ribosomal structure at a sufficiently high resolution remains a great challenge and task to biochemists and crystallographers.

Amino Acid Sequence↗
PubMed1996

Single molecule detection of RNA reporter probes by amplification with Q beta replicase.

The addition of target-specific probe sequences within MDV RNA, an otherwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally placed probe sequences are dramatically affected by short RNA sequences (spacer elements) at the 5' and 3' ends of the probe sequence. For a given probe sequence, the sequences of the flanking spacer elements effected replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal MDV probes were developed that permitted the reproducible, real time, fluorescence detection of a single RNA molecule in less than 25 min through amplification with Q beta replicase. RNA structural analysis of such probes suggested that the spacer elements functioned by allowing the RNA to fold in a way which substantially maintained the tertiary structure of the MDV domain. MDV reporter probes with suitable replication properties were obtained from libraries of RNA molecules in which the probe sequence was flanked by many different spacer elements (generated by random nucleotide synthesis). We demonstrated that this is a general method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single molecule.

Base Sequence↗
PubMed2003

Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus.

Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

Animals↗
PubMed2000

Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid.

Large RNAs fold into complex structures which determine their biological activities. A full understanding of both RNA structure and dynamics will include the description of the pathways by which these structures are formed. Kinetic footprinting [Sclavi, B., et al. (1997) J. Mol. Biol. 266, 144-159] has been shown to be a powerful method for the study of dynamic processes involving RNA. Here we describe the use of a readily available reagent, peroxynitrous acid, as a kinetic footprinting tool for the study of RNA folding. Hydroxyl radicals generated from this reagent were used to footprint the Tetrahymena ribozyme during its magnesium-dependent folding-in agreement with synchroton X-ray footprinting [Sclavi, B., et al. (1998) Science 279, 1940-1943] and oligonucleotide/hybridization cleavage experiments [Zarrinkar, P. P., and Williamson, J. R. (1994) Science 265, 918-924], this work suggests an ordered, hierarchical folding pathway for the ribozyme. Several slow steps in the folding pathway were observed in the peroxynitrous acid footprinting, but none of these corresponded to the rate-determining step of folding. This suggests that the formation of the global, protected structure is followed by one or more slow local rearrangements to yield the final active structure. These studies illustrate the utility of peroxynitrous acid as a reagent for the elucidation of RNA folding pathways and the study of RNA dynamics.

Animals↗
PubMed1998

Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs.

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.

Alfalfa mosaic virus↗
PubMed1987

Raman spectroscopic study of left-handed Z-RNA.

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.

Circular Dichroism↗
PubMed2004

Reduced contact order and RNA folding rates.

We investigated the relationship between RNA structure and folding rates accounting for hierarchical structural formation. Folding rates of two-state folding proteins correlate well with relative contact order, a quantitative measure of the number and sequence distance between tertiary contacts. These proteins do not form stable structures prior to the rate-limiting step. In contrast, most secondary structures are stably formed prior to the rate-limiting step in RNA folding. Accordingly, we introduce "reduced contact order", a metric that reflects only the number of residues available to participate in the conformational search after the formation of secondary structure. Plotting the folding rates and the reduced contact order from ten different RNAs suggests that RNA folding can be divided into two classes. To examine this division, folding rates of circularly permutated isomers are compared for two RNAs, one from each class. Folding rates vary by tenfold for circularly permuted Bacillus subtilis RNase P RNA isomers, whereas folding rates vary by only 1.2-fold for circularly permuted catalytic domains. This difference is likely related to the dissimilar natures of their rate-limiting steps.

Bacillus subtilis↗
PubMed1984

Affinity-purified antibody as a probe of RNA polymerase II subunit structure.

Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.

Animals↗
PubMed1999

Secondary structure mapping of an RNA ligand that has high affinity for the MetJ repressor protein and interference modification analysis of the protein-RNA complex.

The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box.

Bacterial Proteins↗
PubMed1993

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.

Animals↗
PubMed1990

An RNA Holliday junction? Structural and dynamic considerations of the bacteriophage T4 gene 60 interruption.

A novel interrupted gene motif has been reported in which a 50-nucleotide insertion into bacteriophage T4 gene 60 appears to be present in the message at the time of translation, yet it is not translated. We present here a dynamic model for how translation may be occurring in the neighborhood of the interruption. The model involves formation of an RNA structure with similarities to a Holliday junction, followed by migration of the branch point in a strand exchange between message and interruption. The advantage of this model over previous ones is that at no time is a new tRNA required to pair with a discontinuous template.

Amino Acid Sequence↗
PubMed2003

RNA aptamers that bind the nucleocapsid protein contain pseudoknots.

We screened two independent RNA libraries consisting of molecules of 50 nucleotides of random sequence, one of which had additional viral psi-sequences to isolate RNA aptamers that bound to the mature form of the nucleocapsid (NC) protein of Human Immunodeficiency Virus Type-1 (HIV-1). Surface Plasmon Resonance measurements and gel shift assays showed that the RNA aptamers bound with high affinity and specificity. We employed RNase footprinting to characterize the RNA structures and to map their protein binding sites. Most of the selected RNA aptamers contained a plausible pseudoknot in addition to the characteristic stem-loop structure. Moreover, the pseudoknots were part of the NC binding sites. We propose that higher order structures such as pseudoknots may constitute binding motifs for nucleic acid binding proteins, especially for NC protein, which is a nucleic acid chaperone.

Binding Sites↗
PubMed2006

RNA thermometers.

Temperature is an important parameter that free-living cells monitor constantly. The expression of heat-shock, cold-shock and some virulence genes is coordinated in response to temperature changes. Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is a widely used regulatory strategy. RNA thermometers are complex RNA structures that change their conformation in response to temperature. Most, but not all, RNA thermometers are located in the 5'-untranslated region and mask ribosome-binding sites by base pairing at low temperatures. Melting of the structure at increasing temperature permits ribosome access and translation initiation. Different cis-acting RNA thermometers and a trans-acting thermometer will be presented.

Animals↗