RESUMO
BACKGROUND: Cross talk of tumorimmune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumorimmune microenvironment. RESULTS: Here, we re-exploited one transcriptomic dataset to gain insight into tumorimmune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factorAS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. CONCLUSIONS: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy.
Assuntos
Linfócitos T , Neoplasias de Mama Triplo Negativas , Iniciação Traducional da Cadeia Peptídica , Expressão Gênica , Processamento Alternativo , Técnicas de Cultura de Células , Receptor Cross-Talk , Aminoacilação de RNA de Transferência , Transcriptoma , ImunoterapiaRESUMO
tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Cisteína/metabolismo , Serina-tRNA Ligase/metabolismo , Transferases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Escherichia coli/genética , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Cisteína/genética , Serina-tRNA Ligase/genética , Termodinâmica , Aminoacilação de RNA de Transferência/genética , Transferases/genéticaRESUMO
Glutamyl-tRNA (Glu-tRNA(Glu)) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNA(Glu) can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNA(Glu). Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNA(Glu) distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.
Assuntos
Heme/biossíntese , Peróxido de Hidrogênio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Acidithiobacillus/efeitos dos fármacos , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glutamato-tRNA Ligase/antagonistas & inibidores , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Ácido Glutâmico/metabolismo , Aminoacilação de RNA de Transferência/efeitos dos fármacosRESUMO
Glu-tRNA is either bound to elongation factor Tu to enter protein synthesis or is reduced by glutamyl-tRNA reductase (GluTR) in the first step of tetrapyrrole biosynthesis in most bacteria, archaea and in chloroplasts. Acidithiobacillus ferrooxidans, a bacterium that synthesizes a vast amount of heme, contains three genes encoding tRNA(Glu). All tRNA(Glu) species are substrates in vitro of GluRS1 from A. ferrooxidans.Glu-tRNA(3)(Glu), that fulfills the requirements for protein synthesis, is not substrate of GluTR. Therefore, aminoacylation of tRNA(3)(Glu) might contribute to ensure protein synthesis upon high heme demand by an uncoupling of protein and heme biosynthesis.
Assuntos
Acidithiobacillus/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Ácido Glutâmico/metabolismo , Tetrapirróis/biossíntese , Acidithiobacillus/genética , Aldeído Oxirredutases/metabolismo , Genes Bacterianos/fisiologia , Glutamato-tRNA Ligase/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , RNA de Transferência de Ácido Glutâmico/genética , Especificidade por Substrato , Aminoacilação de RNA de TransferênciaRESUMO
1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized directly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of a cDNA clone corresponding to chick alpha(I)procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos.