RESUMO
BACKGROUND: Spliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced. RESULTS: In this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing. CONCLUSIONS: SLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation.
Assuntos
RNA Líder para Processamento/química , Software , Trans-Splicing , Animais , Genômica , Camundongos , RNA-SeqRESUMO
Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.
Assuntos
Caenorhabditis elegans/genética , Nematoides/genética , Splicing de RNA/genética , RNA Líder para Processamento/genética , Animais , Sequência de Bases , Dosagem de Genes , Ontologia Genética , Genoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Líder para Processamento/química , Especificidade da Espécie , Trans-Splicing/genética , Transcriptoma/genéticaRESUMO
The analysis of protein-nucleic acid interactions is essential for biophysics related research. However, simple, rapid, and accurate methods for quantitative analysis of biomolecular interactions are lacking. We herein establish an electrochemical biosensor approach for protein-nucleic acid binding analysis. Nanoparticle based sensors are fabricated by highly-controlled inkjet printing followed by plasma conversion. A novel bioconjugation method is demonstrated as a simple and rapid approach for protein-based biosensor fabrication. As a proof of concept, we analyzed the binding interaction between unwinding protein 1 (UP1) and SL3ESS3 RNA, confirming the accuracy of this nanoparticle based electrochemical biosensor approach with traditional biophysical methods. We further accurately profiled and differentiated a unique binding interaction pattern of multiple G-tract nucleic acid sequences with heterogeneous nuclear ribonucleoprotein H1. Our study provides insights into a potentially universal platform for in vitro biomolecule interaction analysis using a nanoparticle based electrochemical biosensor approach.
Assuntos
Técnicas Biossensoriais , DNA/química , Ouro/química , Ribonucleoproteínas Nucleares Heterogêneas/química , Nanopartículas Metálicas/química , RNA Líder para Processamento/química , Técnicas EletroquímicasRESUMO
RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.
Assuntos
HIV-1/genética , Conformação de Ácido Nucleico , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Viral/genética , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HumanosRESUMO
RNA folding pathways play an important role in various biological processes, such as (i) the hok/sok (host-killing/suppression of killing) system in E. coli to check for sufficient plasmid copy number, (ii) the conformational switch in spliced leader (SL) RNA from Leptomonas collosoma, which controls trans splicing of a portion of the '5 exon, and (iii) riboswitches--portions of the 5' untranslated region of messenger RNA that regulate genes by allostery. Since RNA folding pathways are determined by the energy landscape, we describe a novel algorithm, FFTbor2D, which computes the 2D projection of the energy landscape for a given RNA sequence. Given two metastable secondary structures A, B for a given RNA sequence, FFTbor2D computes the Boltzmann probability p(x, y) = Z(x,y)/Z that a secondary structure has base pair distance x from A and distance y from B. Using polynomial interpolationwith the fast Fourier transform,we compute p(x, y) in O(n(5)) time and O(n(2)) space, which is an improvement over an earlier method, which runs in O(n(7)) time and O(n(4)) space. FFTbor2D has potential applications in synthetic biology, where one might wish to design bistable switches having target metastable structures A, B with favorable pathway kinetics. By inverting the transition probability matrix determined from FFTbor2D output, we show that L. collosoma spliced leader RNA has larger mean first passage time from A to B on the 2D energy landscape, than 97.145% of 20,000 sequences, each having metastable structures A, B. Source code and binaries are freely available for download at http://bioinformatics.bc.edu/clotelab/FFTbor2D. The program FFTbor2D is implemented in C++, with optional OpenMP parallelization primitives.
Assuntos
Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Protozoário/química , Regiões 5' não Traduzidas , Algoritmos , Animais , Análise de Fourier , Cinética , Conceitos Matemáticos , Simulação de Dinâmica Molecular , Splicing de RNA , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Trypanosomatina/química , Trypanosomatina/genética , Trypanosomatina/metabolismoRESUMO
Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.
Assuntos
Mutagênese Insercional , RNA Ribossômico 5S , RNA Líder para Processamento , Trypanosoma/genética , Sequência de Bases , Ordem dos Genes , Genoma de Protozoário , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 5S/química , RNA Líder para Processamento/química , Alinhamento de Sequência , Trypanosoma/classificaçãoRESUMO
Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5' UTRs, the 5'SL. LARP6 binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5'SL of collagen α2(I) mRNA is more stable than the binding to 5'SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5'SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5'SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5'SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis.
Assuntos
Regiões 5' não Traduzidas/genética , Autoantígenos/genética , Colágeno Tipo I/genética , Colágeno/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Autoantígenos/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Células HEK293 , Humanos , Immunoblotting , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Ribonucleoproteínas/metabolismo , Canais de Translocação SEC , Homologia de Sequência de Aminoácidos , Antígeno SS-BRESUMO
Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3' homogeneity. Here, we explored strategies to also ensure 5' homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II 2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5'-sequence heterogeneity in several cases, significant levels of 5' heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5' homogeneity, we tested the suitability of using a small CRISPR RNA stem-loop at the 5' end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5'-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR-SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5'-CRISPR-based strategy can be combined with affinity purification using the 3'-ARiBo tag for quick purification of RNA with both 5' and 3' homogeneity.
Assuntos
Bacteriófago T7/genética , Cromatografia de Afinidade/métodos , RNA Polimerases Dirigidas por DNA/química , Neurospora/genética , RNA Líder para Processamento/isolamento & purificação , RNA Viral/isolamento & purificação , Proteínas Virais/química , Marcadores de Afinidade/química , Bacteriófago T7/química , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , Heterogeneidade Genética , Sequências Repetidas Invertidas , Neurospora/química , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/genética , Regiões Promotoras Genéticas , Clivagem do RNA , Estabilidade de RNA , RNA Catalítico/química , RNA Catalítico/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Thermus thermophilus/química , Thermus thermophilus/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
RNA has many pivotal functions especially in the regulation of gene expression by ncRNAs. Identification of their structure is an important requirement for understanding their function. Structure prediction alone is often insufficient for this task, due to algorithmic problems, parameter inaccuracies, and biological peculiarities. Among the latter, there are base modifications, cotranscriptional folding leading to folding traps, and conformational switching as in the case of riboswitches. All these require more in-depth analysis of the folding space. The major drawback, which all methods have to cope with, is the exponential growth of the folding space. Therefore, methods are often limited in the sequence length they can analyze, or they make use of heuristics, sampling, or abstraction. Our approach adopts the abstraction strategy and remedies some problems of existing methods. We introduce a position-specific abstraction based on helices that we term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, we have implemented this abstraction in the program RNAHeliCes. Furthermore, we developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. We demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.
Assuntos
Dobramento de RNA , RNA/química , Algoritmos , Sequência de Bases , Biologia Computacional , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Protozoário/química , RNA de Protozoário/genética , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , Software , Termodinâmica , Trypanosomatina/química , Trypanosomatina/genéticaRESUMO
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Proteínas de Helminto/química , Iniciação Traducional da Cadeia Peptídica , Análogos de Capuz de RNA/química , Animais , Ascaris suum , Fosfatos de Dinucleosídeos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Helminto/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Líder para Processamento/químicaRESUMO
Trans-splicing is the joining together of portions of two separate pre-mRNA molecules. The two distinct categories of spliceosomal trans-splicing are genic trans-splicing, which joins exons of different pre-mRNA transcripts, and spliced leader (SL) trans-splicing, which involves an exon donated from a specialized SL RNA. Both depend primarily on the same signals and components as cis-splicing. Genic trans-splicing events producing protein-coding mRNAs have been described in a variety of organisms, including Caenorhabditis elegans and Drosophila. In mammalian cells, genic trans-splicing can be associated with cancers and translocations. SL trans-splicing has mainly been studied in nematodes and trypanosomes, but there are now numerous and diverse phyla (including primitive chordates) where this type of trans-splicing has been detected. Such diversity raises questions as to the evolutionary origin of the process. Another intriguing question concerns the function of trans-splicing, as operon resolution can only account for a small proportion of the total amount of SL trans-splicing.
Assuntos
Trans-Splicing/genética , Trans-Splicing/fisiologia , Animais , Sequência de Bases , Evolução Molecular , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon , Filogenia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Spliceossomos/metabolismoRESUMO
The U1A-SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A-SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of â¼100 µs, â¼50 ms, and â¼2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A-SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM-RNA complex.
Assuntos
RNA Líder para Processamento/química , Ribonucleoproteína Nuclear Pequena U1/química , Humanos , Cinética , Modelos Moleculares , Ligação ProteicaRESUMO
The structures attained by RNA molecules depend not only on their sequence but also on environmental parameters such as their temperature. So far, this effect has been largely neglected in bioinformatics studies. Here, we show that structural comparisons can be facilitated and more coherent structural models can be obtained when differences in environmental parameters are taken into account. We re-evaluate the secondary structures of the spliced leader (SL) RNAs from the seven eukaryotic phyla in which SL RNA trans-splicing has been described. Adjusting structure prediction to the natural growth temperatures and considering energetically similar secondary structures, we observe striking similarities among Euglenida, Kinetoplastida, Dinophyceae, Cnidaria, Rotifera, Nematoda, Platyhelminthes, and Tunicata that cannot be explained easily by the independent innovation of SL RNAs in each of these phyla. Supplementary Table is available at http://www.worldscinet.com/jbcb/.
Assuntos
RNA Líder para Processamento/química , RNA Líder para Processamento/genética , Algoritmos , Animais , Sequência de Bases , Biologia Computacional , Eucariotos , Evolução Molecular , Humanos , Modelos Biológicos , Conformação de Ácido Nucleico , Filogenia , Splicing de RNA , Homologia de Sequência do Ácido Nucleico , Temperatura , TermodinâmicaRESUMO
Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5'-end of a small noncoding RNA, the SL RNA, to the 5'-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50-60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50-60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA-only, 5S rRNA-only, and mixed SL RNA-5S rRNA (SL-5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL-5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA-only array was also found in three dinoflagellates, along with two SL-5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL-5S cluster genomic organizations are an "ancient" and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.
Assuntos
Dinoflagellida/genética , RNA de Protozoário/genética , RNA Líder para Processamento/genética , Animais , Sequência de Bases , Dinoflagellida/metabolismo , Dados de Sequência Molecular , RNA de Protozoário/química , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , RNA Líder para Processamento/química , Alinhamento de Sequência , Trans-SplicingRESUMO
Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position -12 (relative to the 5' splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.
Assuntos
Splicing de RNA , Transporte de RNA , RNA de Protozoário/metabolismo , RNA Líder para Processamento/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA de Protozoário/química , RNA de Protozoário/genética , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMO
BACKGROUND: Genomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1) element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1. To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, here we compare the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Prior studies from several groups have employed a variety of constructs and experimental conditions. RESULTS: Although some idiosyncratic differences in the dimerization details were observed, we find unifying principles in the regulation strategies of the three viral RNAs through long- and short-range base pairing interactions. Presentation and efficacy of dimerization through SL1 depends strongly upon the formation or dissolution of the lower stem of SL1 called stem B. SL1 usage may also be down-regulated by long-range interactions involving sequences between SL1 and the first codons of the gag gene. CONCLUSION: Despite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1.
Assuntos
Lentivirus de Primatas/metabolismo , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , RNA Viral/química , RNA Viral/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Dimerização , HIV-1/genética , HIV-1/metabolismo , HIV-2/genética , HIV-2/metabolismo , Humanos , Cinética , Lentivirus de Primatas/classificação , Lentivirus de Primatas/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso , RNA Viral/genética , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismoRESUMO
We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.
Assuntos
Simulação por Computador , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Animais , Sequência de Bases , Cinética , Dados de Sequência Molecular , RNA/genética , RNA Líder para Processamento/química , RNA Líder para Processamento/genética , Reprodutibilidade dos Testes , Termodinâmica , Fatores de Tempo , TrypanosomatinaRESUMO
The SL1 stem-loop is the dimerization initiation site for linking the two copies of the RNA forming the HIV-1 genome. The 26 nucleotides stem contains a defect consisting on a highly conserved G-rich 1-3 asymmetrical internal loop, which is a major site for nucleocapsid protein binding. Several NMR attempts were undertaken to determine the internal loop structure in the SL1 monomer. However, the RNA constructs used in the different studies were largely mutated, in particular with replacement of the nine nucleotides apical loop by a tetraloop, and divergent results were obtained ranging from a rigid structure to a particularly large flexibility. To investigate the reasons for such discrepancies, we used molecular dynamics simulation of the SL1 monomer to probe the effect of mutations on the conformational stability of the internal loop and of the whole stem. It is found that in the wild-type sequence, the internal loop displays conformational variability originating mainly from the nine nucleotides apical loop flexibility that causes large conformational fluctuations (without changing the average structure) in the 7 bp duplex linking the apical and internal loops. The large amplitude atomic motions in the duplex are transmitted to the internal loop in which they induce conformational changes characterized by a labile hydrogen bond network such as G5 successively H-bonded to A29 and G30. On the contrary, with a four nucleotides apical loop, conformational fluctuations in the duplex are reduced by a factor of 2 and are not sufficiently energizing for promoting changes in the internal loop structure at the time scale of the simulations.
Assuntos
Simulação por Computador , HIV-1/química , HIV-1/genética , Modelos Moleculares , RNA Líder para Processamento/química , RNA Viral/química , Pareamento de Bases , Dimerização , Genoma Viral , Ligação de Hidrogênio , Dados de Sequência Molecular , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA Líder para Processamento/genéticaRESUMO
Spliced leader (SL) trans-splicing is a form of mRNA processing originally described in parasitic kinetoplastids. During this reaction, a short RNA sequence is transferred from the 5'-end of an SL transcript to a splice acceptor site on pre-mRNA molecules. Here we report numerous mRNAs from a dinoflagellate, Karenia brevis, which contain an identical leader sequence at their 5'-terminal end. Furthermore, we have isolated a gene from K. brevis encoding a putative SL RNA containing the conserved splice donor site immediately following the leader sequence. A 1,742-bp DNA fragment encoding a K. brevis 5S gene repeat was found to encode the SL RNA gene, as well as a U6 small nuclear RNA (snRNA) gene, and binding sites for the core components of the splicesome (Sm proteins) involved in RNA splicing. Therefore the K. brevis SL RNA appears to be in a genomic arrangement typical of SL genes in a number of species known to mature their mRNAs by trans-splicing. Additionally, we show that the SL gene exists as a stable snRNA and has a predicted secondary structure typical of SL RNAs. The data presented here support the hypothesis that an SL RNA is present in K. brevis and that maturation of a percentage of mRNAs in K. brevis occurs via a trans-splicing process in which a common SL sequence is added to the 5'-end of mature mRNAs. The occurrence of SL trans-splicing in a dinoflagellate extends the known phylogenetic range of this process.
Assuntos
Dinoflagellida/genética , RNA Líder para Processamento/genética , Trans-Splicing , Animais , Sequência de Bases , Northern Blotting , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Líder para Processamento/química , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411-419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1(35) KC model which was submitted to a 31 ns molecular dynamics simulation (MD). Our results allows to describe the internal dynamics of SL1(35) KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.