Human and animal brains are made up of billions of specialized cells called neurons that act together to drive behavior, learning and memory through the mechanisms that we do not fully understand. Neurons communicate with each other through their long cellular extensions that are composed of proteins, produced in the cells from messenger RNAs. The subject of this research is a recently characterized factor, Mov10, that regulates protein production by unwinding messenger RNAs to facilitate their translation into proteins. Mov10 is necessary for early embryonic development across species and for normal neuron development that includes producing the branches that connect neurons. The goals of this work are to identify the RNAs that Mov10 unwinds, to understand how Mov10?s unwinding activity is regulated and to determine how Mov10 participates in the production of the branching that is critical for neuronal function. This project will involve the training of high school students, undergraduates and graduate students in both scientific methodology and in the scientific process, including hypothesis testing. In addition, this work will inform public talks by the PI for presentation to local and state professional groups about scientific discoveries and the importance of supporting basic research.
Bursts of protein translation are required for normal development and neuronal function but it is unknown how this process is regulated. The goal of this research is to explore the key role of RNA unwinding on RNA fate. RNA helicase Mov10 binds in proximity to MicroRNA Recognition Elements (MREs) in 3'UTRs. It unwinds secondary structures in RNA to allow access of the primary effector protein of the miRNA pathway, Argonaute 2, to MREs. Mov10 was shown to bind mRNAs encoding neuronal projection and cytoskeletal proteins in developing brain, suggesting a role for Mov10 in regulating translation in dendrites. The hypothesis being tested is that Mov10 binds RNA G-quadruplexes (GQs), which are stable RNA secondary structures, to modulate Ago2 association to MREs in the 3?UTR. The Mov10-dependent Ago2 target mRNAs will be identified using a brain-specific Mov10 conditional knockout mouse (cko) on which Ago2-enhanced CLIP (eCLIP) will be performed. RNAs whose steady-state expression is Mov10-dependent will be identified by RNA-seq followed by Gene Ontology to identify cytoskeletal RNAs in both WT and Mov10 cko brain. MREs in the target mRNAs will be identified by mutagenesis of the 3'UTRs expressed in a reporter construct. Regulation of Mov10 activity will be examined by testing the hypothesis that phosphorylation of Mov10 controls its helicase activity. The model GQ, iSpinach, will be used to examine how Mov10 phospho-mimics that are either constitutively phosphorylated or constitutively unphosphorylated unwind GQs. It will also be studied how Mov10 regulates neuronal dendrite formation by expanding on the observation that heterozygous loss of Mov10 leads to reduced dendritic arbors in cultured neurons. The hypothesis being tested is that Mov10 regulates Ago2 association with neuron projection and cytoskeleton mRNAs. The Mov10/Ago2-regulated cytoskeletal RNAs that mediate arborization will be identified by introducing candidate genes from which the Ago2-regulatory regions have been removed into Mov10 cko neurons.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
