GSE120405
RNA co-immunoprecipitation coupled to RNA sequencing (RIP-seq) to identify somatic target mRNAs of the TRIM-NHL protein LIN41
We perform RIP-seq experiments with two C. elegans worm lines: i) lin-41(n2914); him-5(e1490) with transgenic expression of a rescuing flag::gfp::lin-41 transgene (Aeschimann et al., Mol Cell, 2017) and ii) him-5(e1490) with transgenic expression of flag::gfp::sart-3 (Rüegger et al., NAR, 2015) as a control. The him-5(e1490) genetic background results in an increased frequency of males in the population. We used an anti-FLAG antibody for the IP and semi-synchronous populations of animals in the L3 and L4 larval stages as samples. The purpose of the experiment was to identify LIN41 mRNA targets in the soma. From the three independent replicates, we determined a set of LIN41-bound mRNAs using edgeR and FDR < 0.05 as a cutoff. This set contained only seven mRNAs, the previously known targets lin-29, mab-10, mab-3 and dmd-3 (Aeschimann et al., Mol Cell, 2017), as well as lin-41 and the unannotated genes F18C5.10 and C31H5.5. We conclude that LIN41 likely only binds to a few somatic mRNA targets. Intersecting this experiment with differential gene expression experiments upon dys-regulation of LIN41 (Aeschimann et al., Mol Cell, 2017) and phenotypic analysis of mutant strains, we further conclude that during larval development, LIN41 likely regulates only a four direct targets, namely lin-29, mab-10, mab-3 and dmd-3. Additionally, the lin-41 mRNA could be directly targeted by LIN41 as well, but the detection of lin-41 mRNA in the IPs may result from the immunoprecipitation of nascent FLAG::GFP::LIN41 protein, still bound to the translating ribosome on its own mRNA. Overall design: We performed RNA co-immunoprecipitations using an anti-FLAG antibody coupled to RNA sequencing. We sampled semi-synchronous populations of animals in the L3 and L4 larval stages of two different genetic backgrounds: i) lin-41(n2914); him-5(e1490) with transgenic expression of a rescuing flag::gfp::lin-41 transgene (Aeschimann et al., Mol Cell, 2017) and ii) him-5(e1490) with transgenic expression of flag::gfp::sart-3 (Rüegger et al., NAR, 2015).