RESUMO
Over the last years, an expanding family of small regulatory RNAs (e.g. microRNAs, siRNAs and piRNAs) was recognized as key players in novel forms of post-transcriptional gene regulation in most eukaryotes. However, the machinery associated with Ago/Dicer-dependent small RNA biogenesis was thought to be either entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, Saccharomyces cerevisiae, Leishmania major and Plasmodium falciparum. Although the biogenesis of small RNAs from non-coding RNAs represent a minor fraction of the normal small RNA transcriptome in eukaryotic cells, they represent the unique small RNA pathways in Trypanosoma cruzi which produce different populations of small RNAs derived from tRNAs, rRNAs, sn/snoRNAs and mRNAs. These small RNAs are secreted included in extracellular vesicles and transferred to other parasites and susceptible mammalian cells. This process represents a novel form of cross-kingdom transfer of genetic material suggesting that secreted vesicles could represent new relevant pieces in life cycle transitions, infectivity and cell-to-cell communication. Here, we provide for the first time a detailed analysis of the small RNA cargo of extracellular vesicles from T. cruzi epimastigotes under nutritional stress conditions compared to the respective intracellular compartment using deep sequencing. Compared with the intracellular compartment, shed extracellular vesicles showed a specific extracellular signature conformed by distinctive patterns of small RNAs derived from rRNA, tRNA, sno/snRNAs and protein coding sequences which evidenced specific secretory small RNA processing pathways.
Assuntos
Vesículas Extracelulares/química , RNA de Protozoário/análise , Trypanosoma cruzi/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA Ribossômico/análise , RNA Ribossômico/genética , RNA Nuclear Pequeno/análise , RNA Nuclear Pequeno/genética , RNA de Transferência/análise , RNA de Transferência/genética , Trypanosoma cruzi/genéticaRESUMO
Our laboratory is interested in post-translational modifications of histone proteins, with studies ranging from identification of novel modifications to functional characterization of these marks. Ultimately, we seek to provide a greater understanding of how histone modifications work together to form a histone code. This code is thought to regulate the recruitment of effector proteins that regulate the diverse functions associated with DNA, including gene transcription and DNA repair. Our recent studies show that RNA polymerase II recruits a variety of chromatinmodifying enzymes that contribute to the disruption, reassembly and maintenance of chromatin structure during the transcription elongation process. One enzyme we have focused on is Set2, which associates with the transcribing polymerase and methylates nucleosomal H3 on lysine 36. H3K36 methylation results in the recruitment of a histone deacetylase complex which functions to prevent inappropriate transcription initiation from occurring within the transcribed regions of genes. I will discuss our recent progress toward understanding how Set2 contributes to the organization and function of chromatin. In addition, I will highlight our progress on a proteomics project that is providing new insights into how readers of the histone code bind their cognate modifications using high-density histone peptide arrays.
Assuntos
Animais , Bovinos , Polimorfismo Genético/genética , RNA Nuclear Pequeno/análise , Expressão Gênica/genéticaRESUMO
Our laboratory is interested in post-translational modifications of histone proteins, with studies ranging from identification of novel modifications to functional characterization of these marks. Ultimately, we seek to provide a greater understanding of how histone modifications work together to form a histone code. This code is thought to regulate the recruitment of effector proteins that regulate the diverse functions associated with DNA, including gene transcription and DNA repair. Our recent studies show that RNA polymerase II recruits a variety of chromatinmodifying enzymes that contribute to the disruption, reassembly and maintenance of chromatin structure during the transcription elongation process. One enzyme we have focused on is Set2, which associates with the transcribing polymerase and methylates nucleosomal H3 on lysine 36. H3K36 methylation results in the recruitment of a histone deacetylase complex which functions to prevent inappropriate transcription initiation from occurring within the transcribed regions of genes. I will discuss our recent progress toward understanding how Set2 contributes to the organization and function of chromatin. In addition, I will highlight our progress on a proteomics project that is providing new insights into how readers of the histone code bind their cognate modifications using high-density histone peptide arrays.(AU)
Assuntos
Animais , Bovinos , Polimorfismo Genético/genética , RNA Nuclear Pequeno/análise , Expressão Gênica/genéticaRESUMO
Immunoprecipitation analysis of total HeLa cells RNA extract byprotein A-Sheparose purified autoantibodies and pCp 32P-3' end labeling RNAs revealed that U1, U2, U4 and U5 snRNAs are related with anti-Sm or U1nRNP autoantibodies, while the hY1, hY3, hY4 and hY5 scRNAs were related to anti-SSA/Ro autoantibodies present in sera of patient with Systemic Lupus Erythematosus. The authors detected molecular snRNAs and scRNAs specificities by autoantibodies in 71 sera, the molecular RNA specificity for anti-Sm (U1, U2, U4 and U5 snRNAs) was present in 39 percent; anti-SSA/Ro sera reacted against scRNAs (hY1, hY3, hY4 and hY5) in 36 percent, then anti-U1nRNP sera recognized U1 snRNA in 13 percent of sera and anti-rRNP related with rRNA were recognized in 8 percent. Twenty-nine SLE sera were RNA negative. A molecular characterization of the autoantibodies in sera from SLE patients may be a useful tool for clinical and laboratory diagnosis of SLE, and the use of autoantibodies es molecular probes allows to continue exploring some basic mechanism of gene expression