The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position þ 6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.

The Silent Sway of Splicing by Synonymous Substitutions

“…However, if position 7 is mutated along with positions 6 or 9, the reduction in exon inclusion is no longer significant. This result reproduces the finding that the A to C mutation at position 7 rescues the C to T mutation at position 6 either through inhibiting the binding of Sam68, or by preventing the binding of another inhibitory protein (33,34). Similar examples of synergistic or compensatory mutations are observed to occur at the Tra2-␤1 (SRSF10) binding site (positions 18 -28), at the 3Ј inhibitory secondary structure (positions [33][34][35][36][37][38][39][40][41][42], and at the 5Ј splice site (21, 23, 32, 35) (Figs.…”

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

“…Cell extracts, cytosol/nuclear fractionations, 23,58 sucrose gradient fractionation of the nuclear compartment 59 and western blot analysis 23 were performed as previously described. Primary antibodies used (1:1000 dilution; overnight at 4 1C) were the following: rabbit anti-ERK2, mouse antiMyc, rabbit anti-SAM68 and goat anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-tubulin, mouse anti-FLAG (Sigma-Aldrich) and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), mouse anti-RNAPII (CTD4H8; Millipore, Billerica, MA, USA) and mouse anti-SND1.…”

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

“…Deletion of this element promoted the skipping of Rps6kb1 exons 6a, 6b, and 6c, thus preventing the expression of Rps6kb1-002. Sam68 is an established regulator of alternative splicing, and it is known to function by directly associating with A/U-rich elements near 5= splice sites (5,10,12,16). The Rps6kb1 intron 6 UAAU UAAA bipartite sequence is recognized by Sam68 with relatively high affinity.…”

“…Sam68 promotes the skipping of exon 7, leading to a nonfunctional SMN2 protein, and it was shown that the inhibition of Sam68 enhanced exon 7 inclusion in endogenous SMN2 and increased survival motor neuron (SMN) levels in SMA patient cells (12). Expanded CGG repeats in the 5= untranslated region (UTR) of the FMR1 gene causes FXTAS, and Sam68 association with these repeats in RNA aggregates blocks it from fulfilling its splicing functions (13).…”