Rbfox Splicing Factors Promote Neuronal Maturation and Axon Initial Segment Assembly

Abstract

Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.

Secondary motifs enable concentration-dependent regulation by Rbfox family proteins

“…We explored the extent to which secondary motifs can account for CLIP peaks not explained by the presence of a primary motif. To do this, we looked for instances of primary, secondary, or both motif types within 50 bases upstream and downstream of the apex of HiTS-CLIP peaks that appeared in both replicates of 3' UTR or intronic HiTS-CLIP data, analyzing two different datasets ( Figure 3e) 8,14 . In both datasets, ~50% of peaks contained a primary motif or both types of motifs (as seen previously 23 ), while another 15-20% of peaks lacked primary motifs but contained two or more secondary motifs.…”

“…The three mammalian paralogs have high sequence identity (94-99%) in their RNAbinding domains (RBDs), substantially overlapping gene targets, and similar activities, making single knockouts difficult to interpret and regulatory targets challenging to define 8,9 . Nonetheless, mouse Rbfox1 central nervous system (CNS) knockouts have seizures and defects in neuronal excitability, while Rbfox2 CNS knockout mice exhibit dramatic defects in cerebellar development 9,10 .…”

“…Recently, it was shown that Rbfox triple knockout mouse embryonic stem cells fail to develop a mature splicing profile when differentiated into ventral motor neurons, underscoring the roles of these proteins in proper neuronal maturation 8 .…”

“…In 2003, RBFOX1 was found to bind the pentanucleotide GCAUG with high affinity to regulate alterative splicing through binding to flanking intronic regions 19 . (U)GCAUG had been established as a dominant signal in computational analyses of neuronal alternative splicing, and Rbfox proteins have since been shown to regulate numerous alternative exons through this motif in a variety of neuronal subtypes 8,14,20,21 . Binding to the GCAUG motif is mediated by canonical and non-canonical interactions between the protein's single RNA recognition motif (RRM) and the RNA 22 .…”

“…Direct binding of Rbfox proteins to these motifs has not been excluded, but if such binding occurs it is not known to have any biological consequence. Almost all studies of Rbfox binding to date have filtered their datasets for the presence of a GCAUG element, discarding many potential binding sites [8][9][10][12][13][14]34 .…”