Resumen de Sam68 and circrna biogenesis in early development
The key to decipher the complexity of biological and pathological processes relies on both coding and non-coding portions of the genome. The non-coding complement includes intronic regions, repeats, linear long RNAs, highly structured tRNAs and rRNAs, small RNAs and, more recently, circular RNAs (circRNAs). The latter became subject of intense research due to their ability to influence gene expression during development. Contrary to the increasing knowledge on circRNA-mediated regulation during brain and heart development, the complex landscape of circRNAs coordinating earlier developmental stages is still far from being characterized. Several RNA Binding Proteins (RBPs) have been shown to be implicated in the biogenesis of circRNAs, especially the Signal Transduction and Activation of RNA (STAR) family members Quaking and Sam68, but whether their contribution is extended to embryonic development is unknown. In order to fill this gap, we have identified the Sam68 protein interactome during different time points of mESCs differentiation. The GO analysis shows that 50% of the whole interactome is composed by RBPs and all steps of RNA metabolism are well represented. We demonstrate by Size Exclusion Chromatography (SEC) that Sam68 strongly interacts with another member of the STAR family (Slm2), suggesting that some of Sam68 activities might rely on cooperation between STAR members.
Since the canonical roles of Sam68 in RNA processing have been widely characterized, we focused on protein partners that could extend Sam68 functions. Indeed, we show an interaction between Sam68 and Ilf2-Ilf3, essential circRNA biogenesis factors and regulators of pluripotency in mESCs. To further investigate our hypothesis, we performed a genome-wide circRNA analysis upon Sam68 KO condition and our results highlights a significant downregulation of circRNAs, suggesting the implication of Sam68 in the regulation of circRNA biogenesis during differentiation.
Moreover, in order to demonstrate the direct involvement of Sam68 in circRNAs formation, we investigated Sam68 binding profile using a bioinformatic tool developed in our laboratory that estimates the binding propensity between protein-RNA pairs and validating the predicted binding propensity by performing a transcriptome-wide mapping of Sam68 mRNA target using iCLIP2.
Our data revealed that Sam68 preferentially binds in intronic regions and that circRNA-forming transcripts are consistently present among its mRNA targets.
In conclusion, our evidences suggest an important contribution for Sam68 in circRNA biogenesis during differentiation, providing a new layer of regulation during embryonic development.
