A Sam68‐dependent alternative splicing program shapes postsynaptic protein complexes

Abstract

Alternative splicing is one of the key mechanisms to increase the diversity of cellular transcriptomes, thereby expanding the coding capacity of the genome. This diversity is of particular importance in the nervous system with its elaborated cellular networks. Sam68, a member of the Signal Transduction Associated RNA-binding (STAR) family of RNA-binding proteins, is expressed in the developing and mature nervous system but its neuronal functions are poorly understood. Here, we perform genome-wide mapping of the Sam68-dependent alternative splicing program in mice. We find that Sam68 is required for the regulation of a set of alternative splicing events in pre-mRNAs encoding several postsynaptic scaffolding molecules that are central to the function of GABAergic and glutamatergic synapses. These components include Collybistin (Arhgef9), Gephyrin (Gphn), and Densin-180 (Lrrc7). Sam68-regulated Lrrc7 variants engage in differential protein interactions with signalling proteins, thus, highlighting a contribution of the Sam68 splicing program to shaping synaptic complexes. These findings suggest an important role for Sam68-dependent alternative splicing in the regulation of synapses in the central nervous system.

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 ablation also affected the alternative inclusion of exon 22 of the syntaxin-binding protein 5-like ( Stxbp5l , alternatively known as tomosyn-2 ) gene ( Fig 1B ), encoding a protein required for normal motor performance and involved in neurotransmission at motor endplates ( Geerts et al, 2015 ). Furthermore, we investigated the regulation of postsynaptic scaffolding molecules that are potential targets of Sam68 and play a central role in synaptic functions or glutamatergic synapses ( Witte et al, 2019 ; Farini et al, 2020 ). Among them, we selected either of the GABAergic post-synaptic proteins collybistin ( Arhgef9 ), gephyrin ( Gphn ), and densin-180 ( Lrrc7 ), the subunits of the glutaminergic AMPA receptor (AMPAR) Gria2 and Gria3 , which mediates the vast majority of fast synaptic transmission in the central nervous system ( Li et al, 2016 ), and of the ATP-sensitive potassium channel SUR2, encoded by the Abcc9 gene (Nichols, 2016) .…”

“…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.…”

Deep Survey of GABAergic Interneurons: Emerging Insights From Gene-Isoform Transcriptomics

“…Similarly, reports on gephyrin splicing factors Nova1 and Nova2 also indicate cell type specificity (Yuan et al, 2018). Intriguingly, alternative splicing of neurexins, gephyrin and collybistin are commonly regulated by Sam68 , which, in contrast to the above factors displays ubiquitous expression (Witte et al, 2018).…”

Cellular Mechanisms Contributing to the Functional Heterogeneity of GABAergic Synapses

“…Splice isoforms of collybistin, a core component of inhibitory synapses was found to control dendritic inhibitory synapse patterning along the proximal-distal axis (de Groot et al, 2017). Collybistin was later identified as a target for alternative splicing by the RNA binding protein Sam68, which was also shown to control splicing of gephyrin mRNA at the C4 splice cassette known to control post-synaptic clustering (Witte et al, 2019). Whether splicing of mRNA coding for inhibitory proteins occurs locally at individual synaptic sites and contributes to synapse-specific protein composition is currently unknown.…”