Small-hairpin RNAs cause target-independent microRNA dysregulation in neurons and elicit global transcriptomic changes

Small hairpin RNAs (shRNAs) allow highly efficient gene knockdown. Here we employed different shRNAs to knock down the reticulon RTN4A/NogoA in primary neurons. Depletion of NogoA correlates with altered synaptic protein composition and spontaneous neurotransmission. However, similar phenotypes are not observed upon genetic deletion of Nogo or its receptors. Step-wise introduction of mismatches in the seed region of shNogoA provides further evidence that synaptic phenotypes are NogoA-independent. RNA sequencing revealed global changes in the neuronal transcriptome of cultures transduced with the original shNogoA or closely related variants. Transcriptomic changes are shRNA seed sequence dependent, but not target-specific. Parallel sequencing of small non-coding RNAs revealed dysregulation of microRNAs. Computational analysis shows that the altered miRNA composition correlates with changes in mRNA expression and preferentially affects protein-protein networks that function at synapses. Thus, off-target effects associated with shRNAs are an inherent property, and in particular, altered miRNA composition needs careful consideration.