The long noncoding RNA NORAD regulates axonal transport

Abstract

Long noncoding RNAs (lncRNAs) have recently emerged as biological cellular regulators in the neural system. The formation and maintenance of neuronal circuits require stable connections between the synapses of long axons. To enable the functionality of such circuits, mRNAs are continuously transported to distal axons for local translation. The precise spatiotemporal patterns of gene expression are afforded by the action of RNA-binding proteins (RBPs), which regulate the intracellular trafficking of target mRNAs to sites of translation in remote axons and dendrites. However, the precise mechanisms by which RBPs are regulated in neurons are not fully understood. Given the mounting evidence suggestive of regulatory roles of lncRNAs in neuronal development, lncRNAs may coordinately regulate RBPs within neurons. The human lncRNA (LINC00657) has been reported to be induced after DNA damage, hence the name "noncoding RNA activated by DNA damage", or NORAD. Interestingly, I found it was consistently and highly expressed in the human neural system. Therefore, I mechanistically characterized the regulatory role of NORAD in the neural system. In chapter 3, I generated NORAD-suppressed human embryonic stem cells (hESCs) and differentiated them into human cerebral organoids. Undifferentiated NORAD-suppressed hESCs exhibited normal colony morphology and pluripotency marker expression, suggesting that NORAD deficiency did not affect stemness. However, cerebral organoids derived from NORAD-suppressed hESCs did not form neural rosettes and displayed abnormal morphological changes, suggesting NORAD might be important for neural development. In chapter 4, I used CRISPR-assisted RNA–protein interaction detection method to identify the candidate protein with direct binding with NORAD. The protein kinesin light chain 1 (KLC1) directly interacted with NORAD and transported various RBPs along microtubules. Consistent with previous reports, the knockdown of KLC1 also exhibited defects in neurite outgrowth. In chapter 5, I demonstrated that NORAD as a scaffold for the RBP SFPQ interacted directly with KLC1. The knockdown of NORAD disrupted the interaction between Kinesin-1 and SFPQ and caused the mislocalization of SFPQ in the axon. Notably, axonal trafficking of SFPQ is necessary for the coassembly of mRNAs required for neurogenesis. Altogether, these data provide evidence that NORAD may orchestrate the spatial gene expression of a newly identified RNA regulon that is essential for axonal viability.