RESUMO
Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Edição de RNA , Trypanosoma brucei brucei/genética , Fluoresceína/química , Corantes Fluorescentes/química , Genoma Mitocondrial , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Uridina Trifosfato/químicaRESUMO
Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.
Assuntos
Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcriptoma/genética , Sítios de Ligação , Escherichia coli/genética , Motivos de Nucleotídeos/genética , Estrutura Secundária de Proteína , RNA Mensageiro/química , Ribonucleases/genética , Ribossomos/genética , Análise de Sequência de RNARESUMO
mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.
Assuntos
RNA Mensageiro/química , RNA Mensageiro/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Ribossomos/genéticaRESUMO
Ribosome profiling is a new emerging technology that uses massively parallel amplification of ribosome-protected fragments and next-generation sequencing to monitor translation in vivo with codon resolution. Studies using this approach provide insightful views on the regulation of translation on a global cell-wide level. In this review, we compare different experimental set-ups and current protocols for sequencing data analysis. Specifically, we review the pitfalls at some experimental steps and highlight the importance of standardized protocol for sample preparation and data processing pipeline, at least for mapping and normalization.
Assuntos
Biossíntese de Proteínas , Ribossomos/química , Ribossomos/metabolismo , Análise de Sequência , Animais , Códon/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.