RESUMO
RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.
Assuntos
Metabolismo Energético/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Sítios de Ligação , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Fases de Leitura Aberta , Polirribossomos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
RNA polymerase II (RNAP-II) synthesizes the m(7)G-capped Spliced Leader (SL) RNA and most protein-coding mRNAs in trypanosomes. RNAP-II recruitment to DNA usually requires a set of transcription factors that make sequence-specific contacts near transcriptional start sites within chromosomes. In trypanosomes, the transcription factor TFIIB is necessary for RNAP-II-dependent SL RNA transcription. However, the trypanosomal TFIIB (tTFIIB) lacks the highly basic DNA binding region normally found in the C-terminal region of TFIIB proteins. To assess the precise pattern of tTFIIB binding within the SL RNA gene locus, as well as within several other loci, we performed chromatin immunoprecipitation/microarray analysis using a tiled gene array with a probe spacing of 10 nucleotides. We found that tTFIIB binds non-randomly within the SL RNA gene locus mainly within a 220-nt long region that straddles the transcription start site. tTFIIB does not bind within the small subunit (SSU) rRNA locus, indicating that trypanosomal TFIIB is not a component of an RNAP-I transcriptional complex. Interestingly, discrete binding sites were observed within the putative promoter regions of two loci on different chromosomes. These data suggest that although trypanosomal TFIIB lacks a highly basic DNA binding region, it nevertheless localizes to discrete regions of chromatin that include the SL RNA gene promoter.