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Transcriptome-wide analysis of HuD (ELAVL4) RNA interactome and post-transcriptional regulation in a motor neuron cell line

ABSTRACT: 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

ORGANISM(S): Mus musculus

PROVIDER: GSE115490 | GEO | 2018/07/13

REPOSITORIES: GEO

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Project description:Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by compromised function of the neuromuscular junction (NMJ), manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterised. Based on the role of MYO9A as a negative regulator of RhoA and an actin-based molecular motor, loss of MYO9A was hypothesised to affect the neuronal cytoskeleton, thus leading to impaired vesicular protein transport within the neuron. MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells) were used to assess the effect on the cytoskeleton revealing altered expression of a number of cytoskeletal proteins important for neuronal cell structure and intracellular transport. Based on these findings, the effect on vesicular protein transport was determined using a vesicular recycling assay revealing impaired recycling of neuronal relevant growth factor receptor. In addition, an unbiased approach utilising proteomic profiling of control and MYO9A-depleted NSC-34 cells was utilised to identify key players of the pathophysiology. Proteomic data support a role for defective vesicular transport and identified affected proteins which are also involved in the manifestation of other neuromuscular disorders. Prompted by the clear indication of perturbed protein transportation, further proteomics-based secretomic analysis of NSC-34 cells have been performed to identify whether secretion is similarly affected. Indeed, this led to the identification of a potential therapeutic target, agrin. Zebrafish lacking MYO9A orthologues (myo9aa/ab) were treated with "Agrin Biologic", an agrin compound, and amelioration of defects in neurite extension and in movement of the zebrafish was observed. Our combined data not only allow new insights into the pathophysiology of CMS and show that loss of MYO9A affects the neuronal cytoskeleton, leading to impaired transport and vesicular recycling of proteins, but on a more general note also represent a successful biomedical approach: from the identification of the underlying pathomechanism to the definition of a therapeutic intervention concept.

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Transcriptome-wide analysis of HuD (ELAVL4) RNA interactome and post-transcriptional regulation in a motor neuron cell line

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