Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain Type-2 immunity [miRNA-seq]

Argonaute (AGO) proteins execute Micro-RNA (miRNA) mediated gene silencing. However it is currently debated whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3 and AGO4) are required for miRNA activity. To address this, we generated a mouse with deleted Ago1, Ago3 and Ago4 genes (Ago134), and found that these Argonaute proteins are completely redundant for miRNA biogenesis, miRNA homeostasis or miRNA function, a role that is carried out exclusively by AGO2. Instead, AGO1/3/4 are required to curtail the expansion of Type-2 adaptive immunity in mice via regulation of precursor-mRNA (pre-mRNA) splicing in CD4+ TH lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing. Our work uncouples AGO1, AGO3 and AGO4 from miRNA-mediated RNAi, discovers a new AGO3:SF3B3 complex in the cellular nucleus, and reveals an underappreciated mechanism by which AGO proteins prevent hyper-inflammatory disease. Comparison of miRNAs from wild type, AGO2 knockout and AGO1/AGO3/AGO4 knockout CD4 T cells. Mouse splenocytes were isolated and naïve CD4+ T cells were obtained via beads purificationa and FACS sorting. Naïve CD4+ T cells were activated and polarized towards Th2 subtype, and total RNA collected at day 0, day 7 and day 14 of in vitro culture. Total RNA was subject to miRNA Seq using TrueSeq Illumina kit. Paired-end sequencing of high depth reads in naïve and activated T cells (nT and TH2 7 days) from littermate WT and AGO134-KO mice, and CD4-Cre;AGO2flox/flox and CD4-Cre mice.