Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNA(Ser) are necessary for pseudouridine (Ψ) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNA(Ser) had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Ψ formation. Deletion analysis showed that the tRNA(Ser) TΨC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TΨC stem-loop exhibited significant Ψ formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TΨC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed.     

Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E. coli tRNA(Asp)

A 77-mer RNA with the sequence of Eschlerichia coli tRNA(Asp) has been chemically synthesised using standard automated phosphoramidite chemistry with the coupling reagent 4,5-dicyanoimidazole (DCI). The synthesis was carried out on a 1000 A CPG-column and. after deprotection and gel purification, a yield of about 7 mmol with a purity of > 95% was reproducibly obtained. By comparing automated synthesis of the 77-mer RNA using 1H-tetrazole and DCI as activator, DCI is advantageous in producing longer RNAs. However, for shorter RNAs ( <40 mer) no difference could be observed. In addition to the all-ribo tRNA(Asp) carrying the wild-type sequence, two variants were synthesised, one with a single C to G48 mutation and the second with a 2'-deoxy modification at C48. The three tRNAs were tested for their aminoacylation efficiency and high affinity binding to E. coli RNase P RNA. The results demonstrate that chemically synthesised 77-mer oligoribonucleotides can be successfully used for structure function studies.     

Common evolutionary trends for SINE RNA structures

Short interspersed elements (SINEs) and long interspersed elements (LINEs) are transposable elements in eukaryotic genomes that mobilize through an RNA intermediate. Understanding their evolution is important because of their impact on the host genome. Most eukaryotic SINEs are ancestrally related to tRNA genes, although the typical tRNA cloverleaf structure is not apparent for most SINE consensus RNAs. Using a cladistic method where RNA structural components were coded as polarized and ordered multistate characters, we showed that related structural motifs are present in most SINE RNAs from mammals, fishes and plants, suggesting common selective constraints imposed at the SINE RNA structural level. Based on these results, we propose a general multistep model for the evolution of tRNA-related SINEs in eukaryotes.     

Flexible zinc finger requirement for binding of the transcriptional activator staf to U6 small nuclear RNA and tRNA(Sec) promoters.

The transactivator Staf, which contains seven zinc finger motifs, exerts its effect on gene expression by binding to specific targets in small nuclear RNA (snRNA) and snRNA-type gene promoters. In this work, binding site selection allowed us to identify the 21-base pair ATTACCCATAATGCATYGCGG sequence as the high affinity consensus binding site for Staf. It shows a high sequence divergence with Staf-responsive elements in the Xenopus selenocysteine tRNA (tRNA(Sec)) and human U6 snRNA promoters. By using a combination of approaches, we analyzed the interaction of wild-type and truncated Staf zinc finger domains with the consensus, Xenopus tRNA(Sec), and human U6 sites. Two main conclusions emerged from our data. First, the data clearly indicate that zinc finger 7 does not establish base-specific contacts in Staf-DNA complexes. The second conclusion concerns zinc finger 1, which is required for the binding to the Xenopus tRNA(Sec) site but is dispensable in the case of the human U6 site. Taking into account the sequence differences in the two sites, these findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible manner, illustrating how a protein can interact with DNAs containing targets of different sequences.     

The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity

The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site.     

The SRP9/14 subunit of the human signal recognition particle binds to a variety of Alu-like RNAs and with higher affinity than its mouse homolog.

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions.     

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved

Summary In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.     

RNA sequence analysis using covariance models.

We describe a general approach to several RNA sequence analysis problems using probabilistic models that flexibly describe the secondary structure and primary sequence consensus of an RNA sequence family. We call these models 'covariance models'. A covariance model of tRNA sequences is an extremely sensitive and discriminative tool for searching for additional tRNAs and tRNA-related sequences in sequence databases. A model can be built automatically from an existing sequence alignment. We also describe an algorithm for learning a model and hence a consensus secondary structure from initially unaligned example sequences and no prior structural information. Models trained on unaligned tRNA examples correctly predict tRNA secondary structure and produce high-quality multiple alignments. The approach may be applied to any family of small RNA sequences.     

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn’s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features.     

ConStruct: a tool for thermodynamic controlled prediction of conserved secondary structure.

A tool for prediction of conserved secondary structure of a set of homologous single-stranded RNAs is presented. For each RNA of the set the structure distribution is calculated and stored in a base pair probability matrix. Gaps, resulting from a multiple sequence alignment of the RNA set, are introduced into the individual probability matrices. These 'aligned' probability matrices are summed up to give a consensus probability matrix emphasizing the conserved structural elements of the RNA set. Because the multiple sequence alignment is independent of any structural constraints, such an alignment may result in introduction of gaps into the homologous probability matrices that disrupt a common consensus structure. By use of its graphical user interface the presented tool allows the removal of such misalignments, which are easily recognized, from the individual probability matrices by optimizing the sequence alignment with respect to a structural alignment. From the consensus probability matrix a consensus structure is extracted, which is viewable in three different graphical representations. The functionality of the tool is demonstrated using a small set of U7 RNAs, which are involved in 3'-end processing of histone mRNA precursors. Supplementary Material lists further results obtained. Advantages and drawbacks of the tool are discussed in comparison to several other algorithms.     

Recognition of duplex DNA by RNA polynucleotides.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence.     

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNA^Phe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNA^Phe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.     

Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P

The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5' leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5' leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (> 900-fold) contributions of the protein subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.     

An essential domain in Saccharomyces cerevisiae U14 snoRNA is absent in vertebrates, but conserved in other yeasts.

U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic precursor rRNA. The U14 RNA from Saccharomyces cerevisiae is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function. This element, known as the Y-domain, is located in the U14 sequence between two universal sequences that base pair with 18S rRNA. Sequence data obtained for the U14 homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to S.cerevisiae. Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight invariant nucleotides. Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction. Several plant U14 RNAs were found to contain similar structures, though with an unrelated consensus sequence in the loop portion. The U14 gene from the most distantly related yeast, Schizosaccharomyces pombe, was found to be active in S.cerevisiae, showing that Y-domain function is conserved and that U14 function can be provided by variants in which the essential elements are embedded in dissimilar flanking sequences. This last result suggests that U14 function may be determined solely by the essential elements.