Structural and Functional Investigation of the Sam68/p300 Protein Complex

Abstract

Sam68 is an RNA-binding protein that is involved in a number of processes related to gene expression, including transcriptional events and pre-mRNA alternative splicing. Although the exact mechanism by which Sam68 exerts its role in transcriptional regulation remains largely unknown, its interaction with multiple transcription factors may enable Sam68 to act as a transcriptional repressor or activator. Previous research discovered a novel complex between Sam68 and transcription factors p300 and CBP. This interaction has been shown to reduce β-catenin-mediated transcription, alter CD44 alternative splicing and is targetable by the ICG-001/CWP family of small molecules. However, there is limited structural and functional information regarding the Sam68/p300 complex. Gathering a wider understanding of how these proteins interact, and their biological significance, is important for comprehending the mechanisms behind Sam68/p300-mediated regulation of transcription and splicing. Through the use of several techniques, the interaction interface was assigned to the CK region of Sam68 and the TAZ2 domain of p300. In vivo splicing assays suggest that p300 enhances Sam68-mediated splicing of Bcl-x and Mcl-1. This contrasts with RNA-Seq data suggesting that overexpression of p300 causes a global reduction in the number of genes undergoing differential alternative splicing by Sam68. Additionally, splicing analysis using RNASeq reveals that Sam68’s role in splicing can be both dependent and independent of transcriptional events. Evaluating the transcriptomic landscape with RNA-Seq provides definitive evidence for Sam68 function in gene expression regulation with events being both dependent and independent of p300. This role of Sam68 in gene regulation is likely to be primarily driven by mechanisms involving lncRNAs. Finally, the relationship between Sam68 and p300 in transcriptional and splicing events requires further investigation due to suboptimal levels of p300 associated with its overexpression. Nonetheless, this research provides additional evidence for the Sam68/p300 complex and has further characterised it both structurally and functionally.</p