Microsatellites that violate Chargaff's second parity rule have base order-dependent asymmetries in the folding energies of complementary DNA strands and may not drive speciation

Models for meiotic recombination based on Crick's “unpairing postulate” require symmetrical extrusion of stem-loop structures from homologous DNA duplexes. The potential for such extrusion is abundant in many species and, for a given single-strand segment, can be quantitated as the “folding of natural sequence” (FONS) energy value. This, in turn, can be decomposed into base order-dependent and base composition-dependent components. The FONS values of top and bottom strands in most Caenorhabditis elegans segments are close, as are the corresponding base order-dependent and base composition-dependent components; any discrepancies are in the base composition-dependent component. This suggests that the strands would extrude with similar kinetics. However, interspersed among these segments and at the ends of chromosomes (telomeres) are segments containing short tandem repeats (microsatellites) which, by virtue of their high variability, have been postulated to inhibit the pairing of homologous chromosomes and hence drive speciation. In these segments, there are usually wide discrepancies between the FONS values of top and bottom strands, mainly attributable to differences in base order-dependent components. Analyses of artificial microsatellites of different unit sizes and base compositions show that this asymmetrical distribution of folding potential is greatest for microsatellites when the units are short and violate Chargaff's second parity rule. It is proposed that when there is folding asymmetry, recombination proceeds by special, strand-biased, somatic mechanisms analogous to those operating with Chi sequences in Escherichia coli. If meiotic recombination in the germ-line requires extrusion symmetry, then a general inhibitory influence of microsatellite-containing segments could mask the antirecombinational influence of their variability. Thus, microsatellites may not have driven speciation.     

Reciprocal relationship between stem-loop potential and substitution density in retroviral quasispecies under positive Darwinian selection

Nucleic acids have the potential to form in trastrand stem-loops if complementary bases are suitably located. Computer analyses of poliovirus and retroviral RNAs have revealed a reciprocal relationship between statistically significant stem-loop potential and sequence variability. The statistically significant stem-loop potential of a nucleic acid segment has been defined as a function of the difference between the folding energy of the natural segment (FONS) and the mean folding energy of a set of randomized (shuffled) versions of the natural segment (FORS-M). Since FONS is dependent on both base composition and base order, whereas FORS-M is solely dependent on base composition (a genomic characteristic), it follows that statistically significant stem-loop potential (FORS-D) is a function of base order (a local characteristic). In retroviral genomes, as in all DNA genomes studied, positive FORS-D values are widely distributed. Thus there have been pressures on base order both to encode specific functions and to encode stem-loops. As in the case of DNA genomes under positive Darwinian selection pressure, in HIV-1 specific function appears to dominate in rapidly evolving regions. Here high sequence variability, expressed as substitution density (not indel density), is associated with negative FORS-D values (impaired base-order-dependent stem-loop potential). This suggests that in these regions HIV-1 genomes are under positive selection pressure by host defenses. The general function of stem-loops is recombination. This is a vital process if, from among members of viral quasispecies, functional genomes are to be salvaged. Thus, for rapidly evolving RNA genomes, it is as important to conserve base-order-dependent stem-loop potential as to conserve other functions.     

The key role for local base order in the generation of multiple forms of China HIV-1 B'/C intersubtype recombinants

Background  

HIV-1 is a retrovirus with high rate of recombination. Increasing experimental studies in vitro indicated that local hairpin structure of RNA was associated with recombination by favoring RT pausing and promoting strand transfer. A method to estimate the potential to form stem-loop structure by calculating the folding of randomized sequence difference (FORS-D) has been used to investigate the relationship between secondary structure and evolutionary pressure in some genome. It showed that gene regions under strong positive "Darwinian" selection were associated with positive FORS-D values. In the present study, the sequences of HIV-1 subtypes B' and C, both of which represent the parent strains of CRF07_BC, CRF08_BC and China URFs, were selected to investigate the relationship between natural recombination and secondary structure by calculating the FORS-D values.     

Stem-loop potential in MHC genes: a new way of evaluating positive Darwinian selection?

The domains of polymorphic major histocompatibility complex (MHC) proteins which interact with peptides and T-cell receptors are considered to have been under positive evolutionary selection pressure. Evidence for this is a high ratio of non-synonymous to synonymous mutations in the corresponding genomic domains. By this criterion snake venom phospholipaseA ₂ genes have also been under positive selection pressure. Recent studies of the latter genes indicate that positive selection has overridden an evolutionary pressure on base order which normally promotes the potential to extrude single-strand stem-loops from supercoiled duplex DNA (fold pressure). This has resulted in base order-dependent stem-loop potential being shifted to introns, which are highly conserved between species. It is now shown that, like snake venom phospholipaseA ₂ genes, the domains of polymorphic MHC genes which appear to have responded to positive selection pressure have decreased base order-dependent stem-loop potential. The evolutionary pressure to generate stem-loop potential (believed to be important for recombination) has been overridden less in exons under positive Darwinian selection. Thus, base order-dependent stem-loop potential shows promise as an independent indicator of positive selection.     

Local Base Order Influences the Origin of ccr5 Deletions Mediated by DNA Slip Replication

CCR5 is a seven-transmembrane G-protein-coupled receptor that binds the CC-chemokines including RANTES, eotaxin, MIP-1α and β. CCR5 serves as an essential coreceptor for cell entry of R5 (macrophage-tropic, nonsyncytium-inducing) strains of HIV-1. To date, four deletions have been found in human and primate ccr5. There is little evidence, however, on how these deletion mutations occur. In the present study, we analyzed ccr5 sequences of both mutants and wild type and found that direct repeats flanked the breakpoints of the deletions, suggesting that these deletions resulted from slipped mispairing during DNA replication. Of particular interest was the location of these deletions in or near the regions with higher negative FORS-D values. High negative FORS-D values stand for high stem-loop potential determined by base order and influence mainly the formation of stem-loop structures. Therefore, the particular location of these deletions suggests that the local sequence of bases might be important in the initiation of deletions mediated by DNA slip replication in concert with direct repeats. Contributed to this paper equally     

Calculation of folding energies of single-stranded nucleic acid sequences: conceptual issues

The stability of a folded single-stranded nucleic acid depends on the composition and order of its constituent bases and may be assessed by taking into account the pairing energies of its constituent dinucleotides. To assess the possible biological significance of a computed structure, Maizel and coworkers in the 1980s compared the energy of folding of a natural single-stranded RNA sequence with the energies of several versions of the same sequence produced by shuffling base order. However, in the 2000s many took as self-evident the view that shuffling at the mononucleotide level (single bases) was conceptual wrong and should be replaced by shuffling at the level of dinucleotides (retaining pairs of adjacent bases). Folding energies then became indistinguishable from those of corresponding shuffled sequences and doubt was cast on the importance of secondary structures. Nevertheless, some continued productively to employ the single base shuffling approach, the justification for which is the topic of this paper. Because dinucleotide pairing energies are needed to calculate structure, it does not follow that shuffling should not disrupt dinucleotides. Base shuffling allows determination of the relative contributions of base composition and base order to total folding energy. The potential for secondary structure arises from pressures acting at both DNA and RNA levels, and is abundant throughout genomes-with a probable primary role in recombination. Within a gene the potential can often be accommodated, and base order and composition work together (values have the same negative sign) in contributing to total folding energy. But sometimes protein-coding pressure on base order conflicts with the pressure for secondary structure and the values have opposite signs. Total folding energy can be deemed of potential biological significance when the average of several readings is significantly less than zero.     

The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Complexes of different electrophoretic mobility containing the stem-loop binding protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone mRNA, are present in both nuclear and cytoplasmic extracts from mammalian cells. We have determined the effect of changes in the loop, in the stem and in the flanking sequences on the affinity of the SLBP for the 3' end of histone mRNA. The sequence of the stem is particularly critical for SLBP binding. Specific sequences both 5' and 3' of the stem-loop are also required for high-affinity binding. Expanding the four base loop by one or two uridines reduced but did not abolish SLBP binding. RNA footprinting experiments show that the flanking sequences on both sides of the stem-loop are critical for efficient binding, but that cleavages in the loop do not abolish binding. Thus all three regions of the RNA sequence contribute to SLBP binding, suggesting that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure recognized by the SLBP.     

The 5′ Stem-Loop From Human U4 snRNA Adopts a Novel Conformation Stabilized by G-C and G-U Base Pairs

Abstract

¹H NMR and molecular modeling studies of the 5′ stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5′ side and a single bulged A on the 3′ side. Both stems also had bulged A residues. ¹H NMR experiments performed on solutions of the 35mer stem-loop, the stem region, and the loop region confirmed the 35mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.     

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.     

The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing.

3' Processing of sea urchin H3 histone pre-mRNA depends on a small nuclear RNP which contains an RNA of nominally 60 nucleotide length, referred to below as U7 RNA. The U7 RNA can be enriched by precipitation of sea urchin U-snRNPs with human systematic lupus erythematosus antiserum of the Sm serotype. We have prepared cDNA clones of U7 RNA and determined by hybridization techniques that this RNA is present in sea urchin eggs at 30-fold lower molar concentration than U1 RNA. The RNA sequences derived from an analysis of eight U7 cDNA clones show neither homologies nor complementarities to any other know U-RNAs. The 3' portion of the presumptive RNA sequence can be folded into a stem-loop structure. The 5'-terminal sequences would be largely unstructured as free RNA. Their most striking feature is their base complementarity to the 3' conserved sequences of histone pre-mRNAs. Six out of nine bases of the conserved CAAGAAAGA sequence of the histone mRNA precursor and 13 out of 16 nucleotides from the conserved palindrome can be base paired with presumptive U7 RNA sequence, suggesting a unique hybrid structure for a processing intermediate formed from histone precursor and U7 RNA.     

Solution structure of a consensus stem-loop D RNA domain that plays important roles in regulating translation and replication in enteroviruses and rhinoviruses

Stem-loop D from the cloverleaf RNA is a highly conserved domain within the 5'-UTR of enteroviruses and rhinoviruses. Interaction between the stem-loop D RNA and the viral 3C or 3CD proteins constitutes an essential feature of a ribonucleoprotein complex that plays a critical role in regulating viral translation and replication. Here we report the solution NMR structure of a 38-nucleotide RNA with a sequence that encompasses the entire stem-loop D domain and corresponds to the consensus sequence found in enteroviruses and rhinoviruses. Sequence variants corresponding to Poliovirus type 1 and Coxsackievirus B3 have virtually the same structure, based on small differences in chemical shifts. A substantial number (136) of (1)H-(13)C one-bond residual dipolar coupling (RDC) values were used in the structure determination in addition to conventional distance and torsion angle restraints. Inclusion of the RDC restraints was essential for achieving well-defined structures, both globally and locally. The structure of the consensus stem-loop D is an elongated A-type helical stem capped by a UACG tetraloop with a wobble UG closing base pair. Three consecutive pyrimidine base pairs (two UU and one CU pair) are present in the middle of the helical stem, creating distinctive local structural features such as a dramatically widened major groove. A dinucleotide bulge is located near the base of the stem. The bulge itself is flexible and not as well defined as the other parts of the molecule, but the flanking base pairs are intact. The peculiar spatial arrangement of the distinctive structural elements implies that they may work synergistically to achieve optimal binding affinity and specificity toward the viral 3C or 3CD proteins.     

Structure of an RNA hairpin from HRV-14.

The 5' noncoding region of the picornaviral genome begins with a cloverleaf which is required for viral replication, due at least in part to an interaction with the viral RNA polymerase as part of a fusion with the predominant viral protease. The necessary region of the cloverleaf has previously been narrowed to a highly conserved stem-loop. The solution structure of a 14-nucleotide RNA hairpin, which is part of the conserved stem-loop from human rhinovirus isotype 14, is presented here. The secondary structure of the hairpin is identical to predictions: a five base pair stem is bounded by a triloop with sequence UAU. However, the fold of the triloop is novel, with stacking of the second loop base onto the closing base pair of the stem, and deviations from A form geometry are introduced into the stem regions bordering the triloop, particularly on the 3' side. These deviations and the associated triloop structure could help to explain the distinct sequence conservation and mutational analysis data observed for the stem region of the hairpin, as compared to a second sequentially similar stem in the intact stem-loop.     

In vitro analysis of the interaction between the FinO protein and FinP antisense RNA of F-like conjugative plasmids

The FinO protein regulates the transfer potential of F-like conjugative plasmids through its interaction with FinP antisense RNA and its target, traJ mRNA. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA in vitro. The FinP secondary structure consists of two stem-loop domains separated by a 4-base spacer and terminated by a 6-base tail. Previous studies suggested FinO bound to the smooth 14-base pair helix of stem-loop II. In this investigation, RNA mobility shift analysis was used to study the interaction between a glutathione S-transferase (GST)-FinO fusion protein and a series of synthetic FinP and traJ mRNA variants. Mutations in 16 of the 28 bases in stem II of FinP that are predicted to disrupt base pairing did not significantly alter the GST-FinO binding affinity. Removal of the single-stranded regions on either side of stem-loop II led to a dramatic decrease in GST-FinO binding to FinP and to the complementary region of the traJ mRNA leader. While no evidence for sequence-specific contacts was found, the results suggest that FinO recognizes the overall shape of the RNA and is influenced by the length of the single-stranded regions flanking the stem-loop.     

Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. II. Evidence that a mRNA stem-loop structure is essential for decoding opal (UGA) as selenocysteine

fdnG, encoding the selenopeptide of Escherichia coli formate dehydrogenase-N, contains an in-frame opal (UGA) codon at amino acid position 196 that directs selenocysteine incorporation. We have identified sequences that contribute to the mRNA context required for decoding this UGA as selenocysteine. We identified a potential stem-loop structure immediately downstream of UGA196 that is comparable in size and structure to a stem-loop predicted to form in fdhF, which encodes the selenopeptide of E. coli formate dehydrogenase-H. Mutational analysis of the fdnG stem-loop structure suggests that it is critical for decoding UGA196 as selenocysteine. Our data indicate that both stability and specific nucleotide sequences of the stem-loop likely contribute to the appropriate mRNA context for selenocysteine incorporation into the fdnG gene product.     

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.     

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.     

RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability.

In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.     

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

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.