The neuronal ELAV-like RNA-binding protein HuD (ELAVL4) plays important roles in multiple post-transcriptional regulatory processes, including RNA stability, transport and translation.
More...The neuronal ELAV-like RNA-binding protein HuD (ELAVL4) plays important roles in multiple post-transcriptional regulatory processes, including RNA stability, transport and translation. Besides its functional role in neuronal plasticity, HuD has been implicated in peripheral axon injury recovery and motor neuronal function. The characterization of HuD specific interactions has always been a challenging task due to the high similarity of sequence and structure with the other members of the ELAVL family, and the lack of specific antibodies. To selectively identify in vivo HuD binding sites, we adapted the CRAC protocol (cross-linking and analysis of cDNAs), originally developed for yeast, to be used with mouse motor neuron NSC-34 cells engineered with inducible tagged HuD. In parallel, to characterize the role of HuD in post-transcriptional regulation, we also profiled the transcriptome (total RNA, RNA-Seq), the translatome (polysomal RNA, POL-Seq) and the alternative polyadenilation (APA, 3’end mRNA sequencing) of HuD induced and control NSC-34 cells.
Keywords: HuD, Elavl4, Y3, Rny3, CRAC, RNA interactome, RNA binding, NSC-34, transcriptome profiling, translatome profiling, RNA-Seq, POL-Seq, motor neuron
Overall design: In this study, NSC-34 cell lines were stably transduced with an inducible vector expressing tagged HuD. To identify the RNA interactome of HuD, the CRAC experiment was performed in biological triplicate, and control experiments with an empty vector were performed in parallel, to filter out any aspecific signal. Transcriptome profiling (total RNA, RNA-Seq) and translatome profiling (polysomal RNA, POL-Seq) were performed in HuD induced and control NSC-34 cells, in biological duplicate. Alternative polyadenilation profiling (APA) was performed by 3’end mRNA sequencing of HuD induced and control NSC-34 cells, in biological triplicate.
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