Examining how TNRC6 binding to its targets changes with the T6B peptide using RIPseq
Prior studies have demonstrated that the T6B peptide can selectively de-repress miRNA targets in non-neuronal cells. However, since neurons may have a different expression profile of miRNA pathway genes as well as other binding partners of TNRC6, we sought to validate the intended function of T6B in neurons. We performed RNA immunoprecipitation of TNRC6 followed by sequencing (TNRC6 RIPseq) in cultured cortical pyramidal neurons transduced with mutant T6B (control) or T6B. We reasoned that TNRC6 would indirectly pull down miRNA targets through its high-affinity binding with AGO in control neurons, but not in the presence of T6B, which outcompetes TNRC6 binding to AGO. Overall design: We cultured cortical pyramidal neurons from E18-18.5 mouse embryos, transduced them with AAV expressing mutant T6B (control) or T6B at day in vitro (DIV) 10 and collected them at DIV14.