RNA Primary Sequence Research Articles

Local Similarities Approximation in DNA Sequences Based on Pairwise Sequence Aligner Algorithm

Sequence alignment is a way of arranging primary sequences of DNA, RNA, or protein to identify regions of similarity. This region may be a consequence of functional, structural, or evolutionary relationships between the sequences. An algorithm is proposed for finding approximate local similarities in DNA sequences (AFALS-N). This algorithm is capable of finding the similarity between two sequences by generating all the possible words in the first sequence, then finding the exact matches in the second sequence. The selection of the obtained results is essential to reduce the number of possible results that in turn reduces the searching time. Results show that the proposed algorithm has reduced the searching time to an average of 20% in regard to PatternHunter algorithm. The objective of this work was evident by maintaining balance between the execution time and the size of seeds and the sensitivity. Improved execution time with 66% of sensitivity are obtained with the same word size as those used in other algorithms.

Power to the people: Does Eterna signal the arrival of a new wave of crowd-sourced projects?

What constitutes a scientist, and do you have to be a professional researcher to perform science? These are questions that have unwittingly been asked in recent years by a number of crowd-sourcing initiatives, and none more so than one of the most recent: Eterna [1]. Following in the footsteps of Foldit [2], Eterna is an online puzzle game that is helping to forward our knowledge of how the primary sequence of RNA molecules determines their structure. Indeed, not only is Eterna doing a job that one would have thought would be restricted to highly-trained scientific researchers and complexly coded algorithms, but it also seems to be doing it better. How does Eterna work? Before we proceed, we would encourage readers to go to the Eterna website, as this will provide a further understanding of the game than we are able to give here. However, to summarise: At the beginning of a typical Eterna round, players are presented with a string of adenine residues and a target conformation. They are then challenged to form the string into the target simply by changing adenines to other nucleotides and thereby taking advantage of the interactions between them. It sounds simple, but it’s not long before you have to start taking account of how the placement of a particular nucleotide will affect its neighbour, as well as the effects of the orientation of each pair. In preparing this article we have only played through some of the simpler levels of the game, but even there the sense of achievement at coaxing a string into the target shape is palpable, and at this level the game hasn’t even played its trump card. Achieve 10,000 points and you earn the right to submit sequences to be synthesised in the laboratory. The developers then feedback on the accuracy of solutions. Further, at 20,000 points the players earn the right to design their own puzzles. Why would the developers go to the trouble of synthesising the designs of players though? Surely the game, though an entertaining diversion, doesn’t warrant this? Well, as alluded to above, game players are discovering previously unknown rules about how the primary sequence of RNA dictates its structure. In fact, players quickly began to outperform algorithms that had been specially designed by experts for the task, and the rules that they have discovered are now being incorporated into the next generation of algorithms. Does this mean that the term 'player’ is no longer appropriate for those who have attained this level? Should we really be starting to refer to them as scientists? In order to investigate this, we were very interested in talking to some of the players to explore their motivation, and what they thought the future of this type of this research might be. We were kindly provided with the email addresses of several of the top game players by the developers of Eterna, and sent them a number of questions related to their experience with the game. An exploration of the responses received is presented below.

Investigation of the sequence constitution of miRNA and siRNA

MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) are small non-coding RNAs that play important regulatory roles in animals and plants. The primary sequences of 9164 miRNAs and 14238 siRNAs were analyzed to determine the occurrence of each nucleotide in specific positions of the sequences. The results show that there are positions in which the composition is not completely random. In general the nucleotide cytosine is underrepresented in both miRNA and siRNA sequences, while the nucleotides uracil and adenine are overrepresented in miRNAs and siRNAs, respectively. Possible implications between these findings and the biological functions of small non-coding RNAs are discussed.Keywords: microRNA, RNA primary sequence, ura

The Solution Structure of the ADAR2 dsRBM-RNA Complex Reveals a Sequence-Specific Readout of the Minor Groove

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

Architecture and secondary structure of an entire HIV-1 RNA genome.

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS, and other serious health threats. Viral replication is regulated at many levels, including using conserved genomic RNA structures. Most potential regulatory elements within viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions. Highly used splice acceptors lie in unstructured motifs and hypervariable regions are sequestered from flanking genome regions by stable insulator helices. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by numerous previously unrecognized regulatory motifs and that extensive RNA structure may constitute an additional level of the genetic code.

FASTR3D: a fast and accurate search tool for similar RNA 3D structures

FASTR3D is a web-based search tool that allows the user to fast and accurately search the PDB database for structurally similar RNAs. Currently, it allows the user to input three types of queries: (i) a PDB code of an RNA tertiary structure (default), optionally with specified residue range, (ii) an RNA secondary structure, optionally with primary sequence, in the dot-bracket notation and (iii) an RNA primary sequence in the FASTA format. In addition, the user can run FASTR3D with specifying additional filtering options: (i) the released date of RNA structures in the PDB database, and (ii) the experimental methods used to determine RNA structures and their least resolutions. In the output page, FASTR3D will show the user-queried RNA molecule, as well as user-specified options, followed by a detailed list of identified structurally similar RNAs. Particularly, when queried with RNA tertiary structures, FASTR3D provides a graphical display to show the structural superposition of the query structure and each of identified structures. FASTR3D is now available online at http://bioalgorithm.life.nctu.edu.tw/FASTR3D/.

Spectrum‐like graphical representation of RNA secondary structure

AbstractWe consider graphical representation for RNA secondary structure by assigning four horizontal lines to the unpaired bases A, C, G, U and four horizontal lines to paired bases A′, C′, G′, U′. The RNA graphical representation based on eight equidistant horizontal lines can be viewed as natural extension of the earlier four‐line graphical representation of DNA (Randić et al., Chem Phys Lett, 2003, 371, 202) to RNA. Numerical characterization of RNA is based on 8 × 8 matrices recording the adjacency of the nucleotide bases in the primary sequence of RNA when RNA is read from 5′ to 3′. The approach is illustrated on a set of nine viral RNAs, reported in the literature. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2009

RNA structure: new messages in translation, replication and disease

The ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs took place between 27 and 29 October 2008, in Baeza, Spain, and was organized by E. Martinez‐Salas, G. Belsham & J. Gomez. ![][1] See Glossary for abbreviations used in this article Since the elucidation of the genetic code approximately 40 years ago, the role of RNA in crucial biological processes has gained increasing attention, and is now one of the fastest developing areas in molecular biology and biochemistry. The advances made in structural studies of transfer RNA (tRNA), ribozymes, riboswitches, the ribosome and viral IRES elements in particular have shown the importance of secondary and tertiary structures in RNA biology, and have significantly changed our understanding of the translational control of gene expression (Fig 1). The World Heritage town of Baeza in Andalucia, Spain, hosted the ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs. About 45 participants met in the beautiful buildings of the Sede Antonio Machado at the Universidad Internacional de Andalucia. Figure 1. Schematic illustration of discreet RNA regulatory elements. The translation of an mRNA is regulated by diverse mechanisms that involve both structural and non‐structural RNA elements, as well as interactions with RNA‐binding proteins. IRESes are found in the 5′ UTR and promote cap‐independent translation. Pseudoknots can be located in the 5′ UTR, the 3′ UTR, or the coding region, and their localization influences their effect on translation; for example, initiation, frameshifting and termination. 3′ UTR structural elements such as CREs, CITEs and SLs often function through long‐range RNA interactions. The miRNA target sites are located in the 3′ UTR. The canonical mRNA features of the cap (m7G) and poly(A) are targets of several regulatory interactions. The … [1]: /embed/graphic-1.gif

A General Definition and Nomenclature for Alternative Splicing Events

Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific “AS code” to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS.

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a-mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.

Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5′ adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5′ and 3′ of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific ‘shifty’ codons.

Conformation of Polypyrimidine Tract Binding Protein in Solution

The polypyrimidine tract binding protein (PTB) is an RNA binding protein that normally functions as a regulator of alternative splicing but can also be recruited to stimulate translation initiation by certain picornaviruses. High-resolution structures of the four RNA recognition motifs (RRMs) that make up PTB have previously been determined by NMR. Here, we have used small-angle X-ray scattering to determine the low-resolution structure of the entire protein. Scattering patterns from full-length PTB and deletion mutants containing all possible sequential combinations of the RRMs were collected. All constructs were found to be monomeric in solution. Ab initio analysis and rigid-body modeling utilizing the high-resolution models of the RRMs yielded a consistent low-resolution model of the spatial organization of domains in PTB. Domains 3 and 4 were found to be in close contact, whereas domains 2 and especially 1 had loose contacts with the rest of the protein.

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system

The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.

A historical perspective on RNA editing: how the peculiar and bizarre became mainstream.

The known examples of RNA editing now encompass a variety of alterations of RNA primary sequence that arise from base modifications, nucleotide insertions or deletions, and nucleotide replacements. Hence, the definition of RNA editing has evolved as new systems have been described. This chapter presents a historical perspective on some of the pivotal discoveries that helped direct the current avenues of research in the field of RNA editing.

Mutational analysis of the predicted secondary RNA structure of the Mason-Pfizer monkey virus packaging signal

The 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5′ untranslated region (5′ UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.

Structural probing and mutagenic analysis of the stem-loop required for Escherichia coli dnaX ribosomal frameshifting: programmed efficiency of 50%

Three elements are crucial for the programmed frameshifting in translation of dnaX mRNA: a Shine-Dalgarno (SD)-like sequence, a double-shift site, and a 3′ structure. The conformation of the mRNA containing these three elements was investigated using chemical and enzymatic probes. The probing data show that the structure is a specific stem-loop. The bottom half of the stem is more stable than the top half of the stem. The function of the stem-loop was further investigated by mutagenic analysis. Reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. Stabilizing the top half of the stem gives increased frameshifting beyond the WT efficiency. The identity of the primary RNA sequence in the stem-loop is unimportant, provided that the overall structure is maintained. The calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. The SD-interaction and the stem-loop element act independently to increase frameshifting in dnaX.

Information capacity of the carbohydrate code

Abstract

The guide RNA database.

The RNA editing process in protozoan parasites is controlled by small RNA molecules known as guide RNAs (gRNAs). The gRNA database is a comprehensive compilation of published guide RNA sequences from eight different kinetoplastid organisms. In addition to the RNA primary sequences, information on the gene localization, the experimental verification of the transcripts, and literature citations are provided. Accessory information includes the secondary structures of fourTrypanosoma bruceigRNAs as well as a computer modelled three dimensional gRNA structure. The database is made available as a hypertext document accessible via the World Wide Web (WWW) or from the authors in a printed form.

The Efficiency of Sendai Virus Genome Replication: The Importance of the RNA Primary Sequence Independent of Terminal Complementarity

From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3′ end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3′/5′ end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identifycis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1–31 of the antigenome 3′ end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.