ABSTRACT
As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in Saccharomyces cerevisiae.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Reproducibility of Results , Binding Sites/genetics , RNA-Binding Proteins/metabolism , RNA/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
This study was aimed to investigate the effect of T cells activated by DCs loaded with whole antigens of U266 cells on the U266 cells survival in vitro. Peripheral blood mononuclear cells were isolated from healthy donor, and adherented on culture plate. Adherent cells were cultured in AIM-V serum-free medium or in RPMI 1640 medium contained 20% fetal bovine serum (FBS), supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate killing rate of U266 cells by T cells activated by DCs loaded with whole antigen of U266 cells. The results showed that DCs derived from peripheral blood mononuclear cells cultured by AIM-V serum-free medium or RPMI 1640 medium containing FBS had similar immunophenotype. T cells activated by DCs loaded with whole antigen of U266 cells or mature DCs might kill U266 cells in a dose-dependent manner. It is concluded, DCs derived from peripheral blood mononuclear cells of healthy donor and loaded with whole antigen of U266 cells can induce anti-myeloma response of T cells in vitro.
