ABSTRACT
We introduce a method for analyzing small interfering RNA (siRNA) genetic screens based entirely on off-target effects. Using a screen for members of the Wnt pathway, we demonstrate that this method identifies known pathway components, some of which are not present in the screening library. This technique can be applied to siRNA screen results retroactively to confirm positives and identify genes missed using conventional methods for on-target gene selection.
Subject(s)
Genome/genetics , Genomics/methods , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/genetics , 3' Untranslated Regions/genetics , Algorithms , Animals , Base Sequence , Computational Biology/methods , Gene Expression Profiling , Gene Library , Humans , Models, Genetic , Reproducibility of Results , Wnt Signaling Pathway/geneticsABSTRACT
Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity.
Subject(s)
Cytomegalovirus Infections/immunology , Exonucleases/genetics , Exonucleases/immunology , Killer Cells, Natural/immunology , MicroRNAs/immunology , Adoptive Transfer , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Exoribonucleases , Homeostasis/immunology , Killer Cells, Natural/cytology , Killer Cells, Natural/virology , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Mutant Strains , MicroRNAs/genetics , NK Cell Lectin-Like Receptor Subfamily A/immunology , NK Cell Lectin-Like Receptor Subfamily A/metabolismABSTRACT
Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.
