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.
Highlights
Around 95% of human genes encode multiple mRNA isoforms with different exon contents, greatly expanding the informational capacity of the genome
Since Sam68 does not control Neurexin2 AS4 exon skipping within the mouse cortex (Figure 1C), Sam68 protein was efficiently cross-linked to the Neurexin2 pre-mRNA (Figure 1B; ∼100-fold enrichment of Neurexin2 precipitation with Sam68 compared with IgG)
Precise measurements using fluorescence polarization (FP) showed that SLM2 and Sam68 have very similar in vitro affinity for the 51 nucleotide UWAA-rich sequence downstream of the Neurexin2 AS4 exon (Table 1). These data indicate that the different splicing activity of SLM2 and Sam68 on Neurexin2 AS4 does not correlate with detectable differences in Sam68 and SLM2 protein-binding levels to the endogenous pre-mRNA
Summary
Around 95% of human genes encode multiple mRNA isoforms with different exon contents, greatly expanding the informational capacity of the genome. Many splicing regulator proteins belong to families of evolutionarily related, similar sister proteins that often, but not always, regulate the same target exons These sister proteins include the STAR proteins SLM2, Sam and SLM1; the Transformer proteins Tra2␣ and Tra2; the Polypyrimidine Tract Binding Proteins PTBP1-3; the Muscleblind proteins MBNL1–3; the Epithelial Specific RNA Splicing regulator proteins ESRP1 and ESRP2; RNA binding Fox homolog proteins RBFOX1–3; TIAL and TIA1; as well as others [5]. Many of these paralogs were created by gene duplications very early in vertebrate evolution. To what extent very similar splicing regulator paralogs might select overlapping versus distinct targets is often poorly understood, as are why such similar paralogs have been maintained often over considerable periods of evolutionary time
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