Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control

Abstract

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein–RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.

Synaptic Neurexin Complexes: A Molecular Code for the Logic of Neural Circuits

“…They are characterized by a central KH-type RNA-binding domain and a C-terminal Sam68 domain, and function broadly in many events of alternative splicing in neuronal and non-neuronal cells (Chawla et al, 2009). Puzzlingly, all STAR-family proteins have been shown to be separately essential for neurexin alternative splicing at SS4 (Iijima et al, 2011 and 2014; Ehrmann et al, 2013; Traunmüller et al, 2016; Danilenko et al, 2017). Of these studies, the best evidence exists for SLM2.…”

“…In vitro experiments suggested that SLM2 and Sam68 can both enhance excision of SS4 in both Nrxn1 and Nrxn3 , but that only SLM2 and not Sam68 can do so in Nrxn2 because the Nrxn2 gene contains fewer binding sites for these factors (Danilenko et al, 2017). Additional mechanisms may contribute.…”

Sam68 splicing regulation contributes to motor unit establishment in the postnatal skeletal muscle

“…Previous work indicated that Sam68 is highly expressed in the motor neurons of the spinal cord ( Pagliarini et al, 2015 ), suggesting an important function of this protein in these cells. Sam68 is known to modulate splicing of several genes encoding for synaptic proteins ( Iijima et al, 2011 ; Danilenko et al, 2017 ; Witte et al, 2019 ; Farini et al, 2020 ). To test whether Sam68 regulates splicing of such synaptic genes in the spinal cord, we selected splicing events from several RNA-sequencing and microarray experiments carried out in Sam68-depleted mouse tissues or cells ( Ehrmann et al, 2008 ; Chawla et al, 2009 ; Iijima et al, 2011 ; Paronetto et al, 2011 ; La Rosa et al, 2016 ; Danilenko et al, 2017 ; Witte et al, 2019 ; Farini et al, 2020 ).…”

“…Sam68 deficiency was also reported to impair motor coordination ( Lukong & Richard, 2008 ) and social behavior ( Farini et al, 2020 ). On the other hand, Sam68 has been involved in the pathogenesis of fragile X-associated tremor/ataxia syndrome ( Sellier et al, 2010 ) and spinal muscular atrophy ( Pedrotti et al, 2010 ; Pagliarini et al, 2015 ), as well as in brain development and function ( Iijima et al, 2011 ; Danilenko et al, 2017 ; Witte et al, 2019 ; Farini et al, 2020 ) through modulation of neuron-specific splicing events.…”

A Sam68‐dependent alternative splicing program shapes postsynaptic protein complexes

“…Much rather, it appears that the cellular context and/or signalling state have important impact on the alternative splicing regulation by these STAR proteins. Notably, previous work highlighted the possibility of differential activities based on heterodimer formation between STAR proteins and additional RNA‐binding proteins (RBPs) (Iijima et al., ) or the density of binding motifs in target mRNAs (Danilenko et al., ). This complexity highlights the challenges of predicting outcomes of alternative splicing regulation based on RBP expression or presence of binding motifs and emphasizes the necessity to interrogate alternative splicing programs using cell type‐specific methods in the future.…”