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Structural investigation of Sam68-driven transcription/splicing coupling

About This PhD Project

Project Description

The identification of 20-25,000 human genes by the human genome project came as a big surprise since the estimated number of human proteins is around 130,000. This discrepancy can only be explained if one single gene can generate many proteins. It then became clear that alternative RNA splicing is a major regulatory event in cells, allowing for the production of many messenger RNAs and proteins from a single gene. This process is highly regulated by RNA binding proteins, called splicing factors, and defects in its regulation lead to a large number of diseases, including cancer, often due to overexpression or mutations of splicing factors.

A typical example is the oncogenic splicing factor Sam68, which is overexpressed in a large number of cancers and whose function in alternative splicing is strongly modulated by signaling pathways (1). However, very little is known about the molecular mechanisms that govern these regulations and modulations.

We have recently revealed the molecular basis of RNA recognition by Sam68 and proposed a model of Sam68’s function in alternative splicing (2). However, several lines of indirect evidence suggest that Sam68 might provide a link between transcription and alternative splicing. In collaboration with Prof. I. Eperon, we have observed that Sam68, unlike numerous splicing factors, does not regulate alternative splicing of its target pre-mRNAs in in vitro splicing assays. This observation is consistent with previous work suggesting that the effects of Sam68 on splicing are, at least in part, mediated by its effects on transcription. Accordingly, interactions between Sam68 and transcription-associated factors have been described and were shown to affect either transcription or alternative splicing events. For example, Sam68 interaction with Brm, a component of the SWI/SNF chromatin remodeling complex, decreases RNA Pol II elongation rate and facilitates the inclusion of the variant alternative exons v5 of CD44 (3); Similarly, the transcription co-activator SND1 binds Sam68 and enhance its splicing activity on CD44 exon v5 (4) and Sam68 act as a transcriptional co-activator through its binding to the Androgen Receptor (AR) transcription factor (5) ; in contrast the binding of the proto-oncogenic transcription factor FBI-1 to Sam68 was shown to inhibit Sam68 function in alternative splicing of Bcl-x (6) and Sam68 can act as a transcription repressor by binding directly the transcription co-activator CBP and preventing its function in gene activation (7). The molecular basis of how these interactions affect either transcription or alternative splicing remains unknown.

This project aims at investigating the structures of Sam68 with its transcription factor partners, using X-ray crystallography and Nuclear Magnetic Resonance (NMR). The structures will then be validated by binding assays and functional assays following site-directed mutagenesis of residues directly involved in the interaction. Finally, we will use these structures to design small molecules that interfere with these interactions. In parallel, we have already developed an in vitro transcription/splicing coupled assay that allows us to investigate the coupling between transcription and splicing in a controlled manner. Using this assay we will investigate at the molecular level the contribution of Sam68 and its transcription factor partners in transcription and splicing.

Techniques that will be undertaken during the project

Expression and purification of proteins from bacterial and mammalian cultures; structure determination using NMR, X-ray crystallography, protein-RNA and protein-protein interactions using Isothermal Titration Calorimetry and Fluorescence Polarization; functional assays using in vitro and in vivo transcription and splicing assays.

Available to UK/EU applicants only
Application information


(1) Bielli et al. Endocr. Relat. Cancer 18, R91–R102 (2011).
(2) Feracci et al. Nat. Comms, 10355 (2016)
(3) Batsché et al. Nat Struct Mol Biol 13, 22–29 (2006).
(4) Capellari et al. Oncogene, 33, 3794-3802 (2014)
(5) Rajan et al. J. Pathol. 215, 67–77 (2008).
(4) Bielli et al. EMBO Rep 15, 419–427 (2014).
(5) Hong et al. Mol Cancer Res 1, 48–55 (2002).

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